Yeasts

Biotechnol Appl Biochem, 1991 Aug, 14(1), 93 - 103
Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase; Cooper JM et al.; Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane . The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid . The pH dependence of the two forms of the enzyme differed . Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated . Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km . The activation energy of immobilised CcP was greater in the presence of both H2O2 {16.6 kJ mol-1} and m-chloroperoxybenzoic acid {37.9 kJ mol-1} than for soluble CcP {11.4 and 23.4 kJ mol-1, respectively} . The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.

Biochim Biophys Acta, 1991 Jul 23, 1089(3), 345 - 51
The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae; Bekkers AC et al.; We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast) . The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase . This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein . Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1 . Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1 . The secreted non-glycosylated prophospholipase A2 species was correctly processed . Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S . cerevisiae.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6112 - 6
Isolation of a metal-activated transcription factor gene from Candida glabrata by complementation in Saccharomyces cerevisiae; Zhou PB et al.; Metal-inducible transcription of metallothionein (MT) genes involves the interaction of metal-responsive trans-acting factors with specific promoter DNA sequence elements . In this report, we present a genetic selection using the baker's yeast, Saccharomyces cerevisiae, to clone a gene from Candida glabrata encoding a metal-activated DNA-binding protein denoted AMT1 . This selection is based on the ability of the AMT1 gene product to activate expression of the C . glabrata MT-I gene in a copper-sensitive S . cerevisiae host strain . DNA-binding studies using AMT1 protein expressed in Escherichia coli demonstrate that AMT1 is activated by copper or silver to bind to both the MT-I and MT-II promoters of C . glabrata . Sequence comparison of AMT1 protein to the S . cerevisiae copper- or silver-activated DNA-binding protein, ACE1, indicates that AMT1 contains the 11 amino terminal cysteine residues known to be critical for the metal-activated DNA-binding activity of ACE1 . In contrast, the carboxyl-terminal portion of AMT1 bears only slight similarity at the primary structure level to the same region of ACE1 known to be important for transcriptional activation . These results suggest that the amino-terminal cysteines, and other conserved residues, play an important role in the ability of AMT1 and ACE1 to sense intracellular copper levels and assume a metal-activated DNA-binding structure.

Biochem J, 1991 Jul 15, 277 ( Pt 2), 335 - 40
The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae); Cronin VB et al.; 1 . The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae) . 2 . Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin . Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods . The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species . 3 . The N-terminus of the enzyme is blocked . Fast-atom-bombardment m.s . was used to identify the blocking group as an acetyl one . 4 . Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms . 5 . Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa . Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem . J . (1991) 273, 5.

Biochimie, 1991 Jul-Aug, 73(7-8), 1027 - 35
Aminoacylation of tRNAs as critical step of protein biosynthesis; Cramer F et al.; Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases . The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles . Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps . Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000 . Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme . A simple hypothetical model of the isoleucine binding site is postulated . Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology . Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA . Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction . The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted.

Eur J Biochem, 1991 Jun 15, 198(3), 607 - 11
113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast; Kofod P et al.; 113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast . NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site . The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases . Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site . The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.

Res Microbiol, 1991 Jun, 142(5), 535 - 9
Development of a pH-controlled fed-batch system for budding yeast; Porro D et al.; In the manufacturing of baker's yeast by aerobic fed-batch systems, continuous assessment of the state of the process is necessary for regulating the flow rate (on/off) for growth medium addition . A new, simple method for the fed-batch yeast process has been developed . It is based on pH changes as a suitable parameter for regulating the feed of fresh concentrated medium in response to metabolic activities of the yeast population . Experimental results have shown that it enables the attaining of high cell density with both high productivity and high yields.

Biokhimiia, 1991 Jun, 56(6), 1123 - 30
{A new method of isolation and a new form of transketolase from baker's yeast}; Tikhomirova NK et al.; A new procedure for the isolation of homogeneous transketolase from baker's yeast based on the use of enzyme-specific antibodies immobilized on a insoluble matrix has been developed . The enzyme yield is 90% of its total content in the original yeast extract . The eluate from the immunocolumn was found to contain a previously unknown form of transketolase which represents an enzyme-RNA complex.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 695 - 9
Cellular gels . Purifying and mapping long DNA molecules; Dear PH et al.; Long DNA molecules of greater than 10(5) bp (0.1 Mbp) are easily broken by pipetting . Therefore, chromosomal DNA is generally isolated after embedding cells in a protective coat of agarose . The embedded DNA can then be cut into long pieces and fractionated on gels using pulsed fields, but these pieces are again easily broken if the resolved DNA molecules are recovered from the gels . We now describe a novel gel matrix, a 'cellular' gel, that permits the recovery of resolved fragments from gels in a form that enables facile manipulation without shear . This facilitates purification and restriction mapping of fragments of 0.1-1.0 Mbp . We illustrate the utility of the method by mapping chromosome III of baker's yeast, which has a length of approximately 0.36 Mbp . This method should facilitate purification and restriction mapping of yeast artificial chromosomes.

Wiad Parazytol, 1991, 37(1), 133 - 6
{Biological observations of allergenic mites Suidasia nesbitti (Acarida, Saproglyphidae)}; Chmielewski W; Suidasia nesbitti specimens for laboratory cultures and for biological studies were isolated from infested fish meal . Rearing conditions: temperature +25 degrees C, relative humidity 85%, food - dried baker's yeast . One day old females and males were paired and placed into separate rearing cages and observations of longevity and oviposition of mites were conducted . Fresh laid eggs were observed and mite development cycle was examined . Mean longevity of male (preimaginal + imaginal periods) amount 84.4 days, female in average 67.0 days . Average egg production of female per its whole life was 172.9 . Life history took in average 14.4 days and natural mortality of various instars was 25% . Frequency of females was slightly higher than frequency of males.

Folia Microbiol (Praha), 1991, 36(5), 468 - 77
The production of extracellular and intracellular free amino acids during aerated fermentation of glucose by baker's yeast (Saccharomyces cerevisiae); Malaney GW et al.; During a study of the effects of a high level of NaCl on the content of free intracellular amino acids in baker's yeast grown in aerated fermentation of glucose it was found (Malaney et al . 1988, 1989; Malaney and Tanner 1988) that 0.6 mol/L exogenous NaCl significantly increased the content of free intracellular citrulline, glutamine, ornithine, arginine and lysine (all basic amino acids) over that observed at zero mol/L exogenous NaCl . (Exogenous is defined as salt added beyond that present in the mineral salts in the culture medium.) This paper describes the production and relative relationships of both extracellular and free intracellular amino acids by S . cerevisiae under conditions of high NaCl content in the growth medium at pH 5 and 32 degrees C . For early culture times (6 h), the production of glutamine, citrulline, valine, isoleucine, ornithine, lysine and histidine were all enhanced by the addition of NaCl . For late times (24 h), except for ornithine, the early-time-enhanced amino acids continued to be enhanced by the addition of NaCl . In addition, the yields of several other amino acids also were increased by exogenous salt at this late time . These include aspartic acid, threonine, glutamic acid, cystine, methionine, tyrosine, phenylalanine and arginine.

Prep Biochem, 1991, 21(2-3), 175 - 85
High-yield extraction and purification of glutathione reductase from baker's yeast; Tsai YC et al.; Glutathione reductase was extracted from toluene-treated baker's yeast cells by a two-stage buffer autolysis method . The yeast cells were treated with toluene for 1 h at 40 degrees C . After removal of the toluene, the cells were then allowed to autolysis in buffer for 72 h at 4 degrees C . The cells were collected and resuspended in buffer . A second stage autolysis was carried out for another 96 h at 4 degrees C . The enzyme was purified to 786-fold from the second stage cell autolysate by using two steps of affinity chromatography with triazine dyes (Yellow H-E4G and Yellow H-E6G) coupled to Sepharose CL-4B . By using this simplified method, 1.44 mg (165 units/mg) of glutathione reductase was obtained from 65 g (wet weight) of yeast cells, equivalent to 80% enzyme recovery.

Enzyme Microb Technol, 1991 Jan, 13(1), 24 - 32
Influence of system and molecular parameters upon fractionation of intracellular proteins from Saccharomyces by aqueous two-phase partition; Huddleston JG et al.; The interaction of molecular characteristics of proteins with the physicochemical properties of PEG-phosphate aqueous two-phase systems has been studied . This has involved characterization of protein molecular weight, charge, and hydrophobicity and study of PEG molecular weight and concentration, phosphate concentration, and pH . System characterization has been conducted in the context of limited stage fractionation procedures for protein recovery from baker's yeast . Results are presented which show that the degree of purification achieved is dependent on macromolecular surface properties rather than system operating conditions . A simple conceptual model of partitioning in PEG-phosphate aqueous two-phase systems is presented which is applicable in the rational design of fractionation procedures and serves to limit the amount of empirical experimentation necessary for the establishment of practical operations.

Eur J Biochem, 1990 Dec 27, 194(3), 879 - 87
Kinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions; Baykov AA et al.; Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme-bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+ . We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules . Catalysis by the E . coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types . Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme-substrate complexes . The distribution of 18O-labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible . The equilibrium constant for this process estimated from direct measurements is 5.0 . The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69 . The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme . In the hydrolytic reaction, enzyme-bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.

J Pharmacobiodyn, 1990 Dec, 13(12), 766 - 71
Production of angiotensin-converting enzyme inhibitors from baker's yeast glyceraldehyde-3-phosphate dehydrogenase; Kohama Y et al.; Angiotensin-converting enzyme (ACE) inhibitors were excised from the molecule of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of baker's yeast by heating at 120 degrees C in 1 M AcOH-20 mM HCl . Three inhibitors were then purified by gel-permeation and reverse-phase chromatographies . One of the yeast ACE inhibitors, YG-3, was GAPDH peptide 79-89 (Pro-Ala-Asn-Leu-Pro-Trp-Gly-Ser-Ser-Asn-Val, IC50:18 microM), and contained the sequence homologous to vertebrate ACE inhibitors (GAPDH peptides 79-86 or 81-88) . Other inhibitors, YG-1 (Gly-His-Lys-Ile-Ala-Thr-Phe-Gln-Glu-Arg, IC50: 0.4 microM) and YG-2 (Gly-Lys-Lys-Ile-Ala-Thr-Tyr-Gln-Glu-Arg, IC50: 2 microM), corresponded to amino acid residues 68-77 in two different forms of yeast GAPDH, respectively . Their sequences were quite different from those of the venom peptide family . YG-1 was the most potent ACE inhibitor among yeast and vertebrate GAPDH peptides excised by acid-limited proteolysis . Thus, yeast GAPDH seems to be an excellent source of naturally occurring ACE inhibitors.

FEBS Lett, 1990 Nov 12, 274(1-2), 27 - 9
A new form of baker's yeast transketolase . An enzyme-RNA complex; Tikhomirova NK et al.; Using an immunosorbent, a new form of transketolase, namely, an enzyme-RNA complex, was isolated from a baker's yeast extract . Spontaneous fission of RNA (or its enzymic hydrolysis by RNase) is accompanied by a sharp increase in the catalytic activity of transketolase, which may be directly related to the enzyme's regulation mechanism.

Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8697 - 701
Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation; Koshy TI et al.; Drosophila melanogaster and rat cytochromes c in which proline-30 was converted to alanine or valine were expressed in a strain of baker's yeast, Saccharomyces cerevisiae, where they sustained aerobic growth . The mutations had no significant effect on the spectra or redox potentials but altered drastically the stability of the bond between the methionine-80 sulfur and the heme iron, as judged by four criteria: (i) the alkaline pKa values of the 695-nm band of the ferric form of the mutant proteins decreased by almost 1 pH unit as compared to the wild types; (ii) the acid pKa values increased by 0.5 to 1.2 pH units; (iii) the 695-nm band half-disappeared at temperatures 10-20 degrees C lower in the mutant proteins than in the wild types; and (iv) the 695-nm band of the mutant proteins was susceptible to concentrations of urea that had little influence on their overall structure . The valine-substituted rat cytochrome c had properties intermediate between those of the wild type and the alanine mutant . The destabilized coordinative bond is located in space a long distance from the mutation site . It is suggested that the mutations weaken the hydrogen bond between the carbonyl of residue 30 and the imino group of the imidazole of histidine-18, modifying the bonding of the heme iron by that imidazole, which, in turn, through a trans effect, weakens the bond between the heme iron and the other axial ligand, the sulfur of methionine-80 . Alternatively, the effect of the mutations may be propagated allosterically along the peptide chain.

Bioessays, 1990 Nov, 12(11), 519 - 26
Baker's yeast, the new work horse in protein synthesis studies: analyzing eukaryotic translation initiation; Linder P et al.; The possibility of combining powerful genetic methods with biochemical analysis has made baker's yeast Saccharomyces cerevisiae the organism of choice to study the complex process of translation initiation in eukaryotes . Several new initiation factor genes and interactions between components of the translational machinery that were not predicted by current models have been revealed by genetic analysis of extragenic suppressors of translational initiation mutants . In addition, a yeast cell-free translation system has been developed that allows in vivo phenotypes to be correlated with in vitro biochemical activities . We summarize here the current view of yeast translational initiation obtained by these approaches.

Biochem Int, 1990 Oct, 22(1), 31 - 6
Purification of transketolase from baker's yeast by an immunoadsorbent; Tikhomirova NK et al.; Baker's yeast transketolase has been purified by immunoaffinity chromatography on specific TK antibodies covalently linked to CNBr-agarose . Affinity chromatography allows a 480-fold purification of TK from yeast extracts and about 80% recovery of the original activity . The isolated enzyme has a specific activity of 12-60 U/mg and during polyacrylamide gel electrophoresis performed at pH 8.9 migrates as two protein bands possessing a transketolase activity which corresponds to two isoforms of the enzyme.

Yeast, 1990 Sep-Oct, 6(5), 429 - 40
Characterization of the cytochrome c gene from the starch-fermenting yeast Schwanniomyces occidentalis and its expression in Baker's yeast; Amegadzie BY et al.; A cytochrome c protein gene, CYC10, of the dextran- and starch-fermenting yeast, Schwanniomyces occidentalis was cloned and characterized . The DNA sequence was determined, and the predicted amino acid sequence of the protein-coding region shares close homologies to the cytochrome c genes . A S . occidentalis strain with a disruption of the gene revealed that CYC10 was the only functional cytochrome c protein-encoding gene in S . occidentalis, unlike the two cytochrome c protein genes (CYC1 and CYC7) in Saccharomyces cerevisiae . The CYC10 gene was oxygen-induced but not subject to catabolite repression . The expression of the CYC10 gene was studied in the heterologous yeast S . cerevisiae . The oxygen induction of the gene was found to be identical to that of the CYC1 gene, indicating that these two genes share similar or closely related cis- and trans-acting oxygen regulatory elements . However, the CYC10 gene was glucose repressed in S . cerevisiae strains; a phenomenon which was not observed in the native S . occidentalis cells . Search in the 5' untranslated region of the CYC10 gene revealed some homologies at -425 to -405 to UAS1 of the S . cerevisiae CYC1 gene . A deletion of a segment of upstream region including this sequence abolished expression in S . cerevisiae . Finally the phylogenetic relationships of different yeasts and fungi were determined based upon the amino acid sequences of the cytochrome c proteins . These relationships do not completely agree with classical divisions.

Yeast, 1990 Sep-Oct, 6(5), 403 - 10
Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA; Hardy CM et al.; The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported . Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene . A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA . This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1 . In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene . Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K . lactis, but is not present upstream of other genes . Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K . lactis is closely related to that of Saccharomyces cerevisiae.

Appl Microbiol Biotechnol, 1990 Sep, 33(6), 629 - 32
Properties and repeated use of a reversibly soluble-insoluble yeast lytic enzyme; Taniguchi M et al.; A yeast lytic enzyme was covalently immobilized on an enteric coating polymer, Eudragit S, that is reversibly soluble and insoluble (S-IS) depending on the pH of the reaction medium . The yeast lytic enzyme immobilized on Eudragit S (Y-E) showed a sharp response of solubility to slight changes in pH without decrease in enzymatic activity . The specific activity per amount of enzyme protein of Y-E for dry yeast cells was about two-thirds that of the native enzyme . In both lysis reactions of dry and pressed baker's yeast cells, changing the pH of the reaction medium from 7.0 to 4.8 at an appropriate interval allows the insoluble Y-E and the reaction products (soluble protein for dry yeast cells and invertase and soluble protein for pressed baker's yeast cells) to be repeatedly separated . The reaction method using a reversible S-IS enzyme is a promising procedure for repeated use of the enzyme in a heterogeneous reaction system containing yeast cells as a substrate.

Biochem Int, 1990 Aug, 21(5), 915 - 21
Enhanced proteolysis of glucose-6-phosphate dehydrogenase in the presence of palmitoyl coenzyme A; Orstan A et al.; Palmitoyl coenzyme A at concentrations below its critical micelle concentration increases the rate of proteolysis of baker's yeast glucose-6-phosphate dehydrogenase by proteinase A in the pH range 4-5 . both glucose-6-phosphate and NADP protect glucose-6-phosphate dehydrogenase against proteolysis, but these protective effects are diminished in the presence of palmitoyl coenzyme A . Since palmitoyl coenzyme A is known to dissociate glucose-6-phosphate dehydrogenase into dimers, the results imply that the in vivo half life of glucose-6-phosphate dehydrogenase may be controlled by a process based on the regulation of the oligomeric structure of the enzyme by the collective actions of various molecules, including palmitoyl coenzyme A.

Biotechnol Appl Biochem, 1990 Aug, 12(4), 364 - 9
Medium scale production of L-myo-inositol 1-phosphate; Feth F et al.; L-myo-Inositol-1-phosphate synthetase was purified from baker's yeast, grown in a fermenter in an inositol-deficient medium and analyzed using a new HPLC assay for inositol . This enzyme was used in a procedure, developed from methods partially described in the literature, for the medium scale production and purification of L-myo-inositol 1-phosphate . The identity and purity of the product were confirmed by 1H and 31P NMR spectroscopy.

Eur J Biochem, 1990 Jul 20, 191(1), 123 - 9
Valyl-tRNA synthetase from yeast . Discrimination between 20 amino acids in aminoacylation of tRNA(Val)-C-C-A and tRNA(Val)-C-C-A(3'NH2); Freist W et al.; For discrimination between valine and the 19 naturally occurring noncognate amino acids, as well as between valine and 2-amino-isobutyric acid by valyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values in aminoacylation of tRNA(Val)-C-C-A . The lowest values were found for Trp, Ser, Cys, Lys, Met and Thr (D = 90-870), showing that valine is 90-870 times more frequently attached to tRNA(Val)-C-C-A than the noncognate amino acids at the same amino acid concentrations . The other amino acids exhibit D values between 1,100 and 6200 . Generally, valyl-tRNA synthetase is considerably less specific than isoleucyl-tRNA synthetase, but this may be partly compensated in the cell by valine concentrations higher than those of noncognate acids . In aminoacylation of tRNA(Val)-C-C-A(3'NH2) discrimination factors D1 are in the range of 40-1260 . From D1 values and AMP formation stoichiometry, pretransfer proof-reading factors pi 1 were determined: post-transfer proof-reading factors II 2 were determined from D values and AMP formation stoichiometry in acylation of tRNA(Val)-C-C-A . II 1 values (7-168) show that pretransfer proof-reading is the main correction step, post-transfer proof-reading (II 2 approximately 1-7) is less effective and in some cases negligible . Initial discrimination factors were calculated from discrimination and proof-reading factors according to a two-step binding process . These factors, due to different Gibbs free energies of binding can be related to hydrophobic interaction forces, and a hypothetical 'stopper' model of the amino-acid-binding site is discussed.

Biochem Biophys Res Commun, 1990 Jul 16, 170(1), 182 - 6
The isolation of biologically active mating pheromone, a-factor, from the yeast, Saccharomyces cerevisiae; Proteau G et al.; Haploid cell-types of baker's yeast, Saccharomyces cerevisiae, secrete pheromones which are essential for conjugation . Recently a putative structure for the elusive a-factor pheromone has been reported . In this report we present a procedure to obtain a-factor from batch cultures of cells using hydrophobic Amberlite XAD-2 resin in the growth medium with subsequent differential washings of the resin with organic solvents . We have determined the biological activity of the a-factor preparation by verifying that there is an increase in transcription of the a-factor receptor gene, STE3, by Northern analysis of STE3 mRNA before and after exposure of the appropriate cell type to a-factor . Furthermore, a beta-galactosidase assay of the putative receptor gene fused to the lacZ gene, coding for beta-galactosidase (STE3-lacZ), was done to quantify the biological activity of the a-factor.

Br J Haematol, 1990 Jul, 75(3), 333 - 9
In vivo priming of human normal neutrophils by granulocyte-macrophage colony stimulating factor: effect on the production of platelet activating factor; Aglietta M et al.; The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) (recombinant, mammalian, glycosylated, Sandoz, Schering Plough; 4 micrograms/kg every 12 h for 3 d, s.c.) on platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn glycero-3 phosphorylcholine) production from neutrophils was studied in five cancer patients with normal haemopoiesis . Peripheral blood counts, PAF production and lyso-PAF: acetyl transferase (EC 2.3.1.67) (AT) activity in neutrophils were evaluated before treatment, during treatment and 3 d after treatment had been discontinued . GM-CSF induced a three-fold increase in the number of circulating neutrophils . Neutrophils obtained during treatment produced about twice as much PAF than before treatment in response to a variety of stimuli (N-formyl-methionyl-leucyl-phenylalanine, tumour necrosis factor-alpha, phagocytosis of baker's yeast spores opsonized with C3b) . This increased PAF synthesis and release is concomitant with a 2-3-fold increase in AT activity . Moreover, lower concentrations of stimuli are sufficient to induce PAF synthesis from neutrophils obtained during GM-CSF treatment . Three days after treatment had been discontinued, stimulus induced PAF production had returned to baseline levels . Since GM-CSF induces a marked shift to the left in the Arneth score, the increased PAF release might have been due to the presence of younger granulocytes . This was, however, ruled out by experiments showing that normal neutrophils primed in vitro with GM-CSF produce more PAF when challenged with the same stimuli . The potential relevance of this effect of GM-CSF treatment lies on the crucial role of PAF in inflammatory reactions and its intervention in some immune reactions, including delayed hypersensitivity, and in endotoxic shock . Lastly, increased PAF production from neutrophils may explain some toxicities observed during treatment with high doses of GM-CSF.

Exp Eye Res, 1990 Jun, 50(6), 751 - 7
S-antigen: from gene to autoimmune uveitis; Shinohara T et al.; Retinal S-antigen (S-Ag) is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals . EAU may serve as an animal model for studying human uveitis . As a first step we have determined the nucleotide sequence of an S-Ag gene and its cDNAs . The amino acid sequences were deduced from the cDNAs of various animals and human . Four uveitopathogenic sites in bovine S-Ag were characterized . One of the sites (peptide M) has sequence homology with non-self proteins from baker's yeast, potato, E . coli, hepatitis B virus, moloney murine leukemia virus, Moloney murine sarcoma virus, AKR murine leukemia virus and baboon endogenous virus . Mononuclear cells from animals immunized with peptide M showed significant proliferation when incubated with synthetic peptides corresponding to the amino acid sequences of the above-mentioned foreign proteins . In addition, all the peptides induced EAU in Lewis rats with a dose of 10-2000 micrograms . Moreover, native histone H3 from baker's yeast histone H3 induced EAU in Lewis rats . Thus, we found several examples of antigenic mimicry between self and non-self proteins . These findings establish a base to study further the mechanism of autoimmune inflammation.

Arch Biochem Biophys, 1990 May 15, 279(1), 146 - 50
Inhibition of platelet aggregation by S-(1,2-dicarboxyethyl)glutathione, intrinsic tripeptide in liver, heart, and lens; Tsuboi S et al.; S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) found in animal tissues or baker's yeast showed strong inhibitory effects on blood coagulation and platelet aggregation . The inhibitory effect of blood coagulation was almost the same as those of EDTA, oxalate, and citrate . DCE-GS did not show chelating activity . As for ADP- or thrombin-induced platelet aggregations, DCE-GS exerted a potent effect on the secondary aggregation, while it was less active in the primary aggregation . DCE-GS gave a distinct lag period in the time course of the secondary aggregation induced by collagen and inhibited most strongly the aggregation induced by arachidonic acid compared with those elicited by ADP, thrombin, and collagen . The peptide, however, did not inhibit the platelet aggregation induced by 12-O-tetradecanoylphorbol-13-acetate . Although both DCE-GS and EDTA inhibited the platelet aggregation which was triggered by ADP, their inhibitory manners were entirely different.

Nucleic Acids Res, 1990 May 11, 18(9), 2589 - 97
Solution conformation of several free tRNALeu species from bean, yeast and Escherichia coli and interaction of these tRNAs with bean cytoplasmic Leucyl-tRNA synthetase . A phosphate alkylation study with ethylnitrosourea; Dietrich A et al.; The solution conformation of eight leucine tRNAs from Phaseolus vulgaris, baker's yeast and Escherichia coli, characterized by long variable regions, and the interaction of four of them with bean cytoplasmic leucyl-tRNA synthetase were studied by phosphate mapping with ethylnitrosourea . Phosphate reactivities in the variable regions agree with the existence of RNA helices closed by miniloops . At the junction of these regions with the T-stem, phosphate 48 is strongly protected, in contrast to small variable region tRNAs where P49 is protected . The constant protection of P22 is another characteristics of leucine tRNAs . Conformational differences between leucine isoacceptors concern the anticodon region, the D-arm and the variable region . In several parts of free tRNALeu species, e.g . in the T-loop, phosphate reactivities are similar to those found in tRNAs of other specificities, indicating conformational similarities among tRNAs . Phosphate alkylation of four leucine tRNAs complexed to leucyl-tRNA synthetase indicates that the 3'-side of the anticodon stem, the D-stem and the hinge region between the anticodon and D-stems are in contact with the plant enzyme.

Rev Infect Dis, 1990 May-Jun, 12(3), 406 - 11
Invasive infection with Saccharomyces cerevisiae: report of three cases and review; Aucott JN et al.; Saccharomyces cerevisiae (brewer's or baker's yeast) is a common colonizer of human mucosal surfaces, but its role as a clinically important pathogen has been unclear . We report three cases of life-threatening invasive infection with S . cerevisiae resulting in pneumonia, liver abscess and sepsis, and disseminated infection with cardiac tamponade, respectively . A review of the English-language literature reveals 14 other cases of saccharomyces infection in humans . Severe immunosuppression, prolonged hospitalization, prior antibiotic therapy, and/or prosthetic cardiac valves are the settings where saccharomyces infection has been observed . Because Saccharomyces can be a common saprophytic contaminant, biopsy and pathologic confirmation of infection are often necessary for a definitive diagnosis . Amphotericin B is the treatment of choice for serious infections with this organism.

Proc Natl Acad Sci U S A, 1990 May, 87(9), 3368 - 72
Revision of the oligosaccharide structures of yeast carboxypeptidase Y; Ballou L et al.; The N-linked oligosaccharides from baker's yeast carboxypeptidase Y were analyzed by 1H NMR and specific mannosidase digestion and found to be identical to those from the Saccharomyces cerevisiae mnn9 mutant bulk mannoprotein . The results support the view that the mnn mutants make oligosaccharides that are a true reflection of the normal biosynthetic pathway and confirm that a recently revised yeast oligosaccharide structure is applicable to wild-type mannoproteins.

Chem Phys Lipids, 1990 Apr, 54(1), 43 - 8
Strategies for the chemoenzymatic preparation of optically active 1-alkyn-3-ols; Glanzer BI et al.; A series of (R)- and (S)-1-alkyn-3-ols, chiral building units for the synthesis of leukotrienes and pheromones, were prepared via enantioselective hydrolysis of their racemic esters . While the majority of biocatalysts employed (lipases, fermenting or freeze-dried microorganisms) failed in discriminating between enantiomers, lyophilized cells of baker's yeast (Saccharomyces cerevisiae Hansen) gave (S)-1-alkyn-3-ols and their corresponding (R)-esters with greater than 90% e.e.

Eur J Biochem, 1990 Mar 30, 188(3), 697 - 703
A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract; Eschrich K et al.; The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described . The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases . The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate . In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved . When substrate is supplied continuously, only stable stationary states can be observed . With transient perturbations of the substrate supply, multiple stationary states appear . Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply . A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed . The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6-bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence . The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.

Vopr Pitan, 1990 Mar-Apr, (2), 69 - 74
{The metabolic efficiency of baker's yeast in an atherogenic diet}; Zhikhar LIu et al.; The effect of bakery compressed yeasts in the amounts of 6 and 12 g/100 g of the ration was studied in rats . It was revealed that 12 g of yeasts per 100 g of the atherogenic ration produced a hypocholesterolemic effect . A negative effect of this amount of the yeasts was manifest in the slow growth of the rats' body mass and in an increased ratio of the kidney/body mass . The yeasts amounts used did not protect the test animals from the kidney infiltration with lipids and cholesterol; 12 g of yeasts per 100 g of the ration promoted elevation of sialic acid content in the blood plasma . Morphologic changes were observed only in the aortal wall, they were characteristic of the prelipid stage of atherosclerosis and did not depend on the yeasts amount.

Chem Pharm Bull (Tokyo), 1990 Mar, 38(3), 807 - 9
Protective effect of baker's yeast mannan against Listeria monocytogenes and Pseudomonas aeruginosa infection in mice; Kobayashi M et al.; The neutral mannan (WNM) and the acidic mannan (WAM025) fractions from baker's yeast (Saccharomyces cerevisiae) were found to manifest significant protective effects against intraperitoneal and intravenous infections of Listeria monocytogenes and Pseudomonas aeruginosa in mice . A remarkable decrease in the number of microbial cells in spleen and liver was observed in mice inoculated with these microorganisms after administration of either mannan fraction . In order to clarify the mechanism of the protective effects, we investigated in vitro the bactericidal activity and lysosomal enzyme activities such as myeloperoxidase, acid phosphatase, and neutral protease, in Kupffer cells (KCs) from mice pretreated with either mannan fraction . KCs from mice administered with these mannan fractions showed an enhanced killing effect on these bacteria in vitro, and neutral protease activity was considered to be one of the important factors in the killing effect on both L . monocytogenes and P . aeruginosa.

Eur J Biochem, 1990 Feb 22, 188(1), 165 - 74
Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast; Heidlas J et al.; The NADPH-linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enantioselective reduction of diacetyl (2,3-butanedione) to (S)- and (R)-acetoin (3-hydroxy-2-butanone) {so-called (S)- and (R)-diacetyl reductases} (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity . The clean-up procedures comprising streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B column chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120-fold and 368-fold purifications, respectively . The relative molecular mass of the (R)-diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36,000 . The (R)-enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5-, C6-2,3-diketones, 1,2-cyclohexanedione, 2-oxo aldehydes and short-chain 2- and 3-oxo esters as substrates . The enzyme was characterized by high enantioselectivity and regiospecificity . The Km values for diacetyl and 2,3-pentanedione were determined as 2.0 mM . The Mr of the (S)-diacetyl reductase was determined as 75,000 by means of HPLC filtration of Zorbax GF 250 . The enzyme decomposed into subunits of Mr 48,000 and 24,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The optimum pH was 6.9 . The purified (S)-enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3-, 2,4- and 2,5-diketones, 2-oxo aldehydes, 1,2-cyclohexanedione and methyl ketones as well as 3-, 4- and 5-oxo esters . The 2,3- and 2,4-diketones are transformed to the corresponding (S)-2-hydroxy ketones; 2,5-hexanedione, however, was reduced to (S,S)-2,5-hexanediol . The Km values for diacetyl and 2,3-pentanedione were estimated as 2.3 and 1.5 mM, respectively . Further characterization of the (S)-diacetyl reductase revealed that it is identical with the so-called '(S)-enzyme', involved in the enantioselective reduction of 3-, 4- and 5-oxo esters in baker's yeast.

J Biol Chem, 1990 Jan 15, 265(2), 801 - 7
Characterization of a nonglycosylated single chain urinary plasminogen activator secreted from yeast; Melnick LM et al.; Using site-directed mutagenesis, we have changed the asparagine in human single-chain urinary plasminogen activator (u-PA) at position 302 to an alanine . This alteration removes the only known amino acid residue glycosylated in the protein . The single-chain u-PA containing an alanine residue at position 302 instead of asparagine (scu-PA(N302A} cDNA gene was expressed in the yeast Saccharomyces cerevisiae . Secretion of the protein product into the culture broth was achieved by replacing the human secretion signal codons with those from yeast invertase, adding a yeast promoter from the constitutively expressed glycolytic genes triosephosphate isomerase or phosphoglycerate kinase, and integrating multiple copies of these transcriptional units into the genome of yeast strains carrying the "supersecreting" mutation ssc1 . When fermented in a fed-batch mode, these recombinant baker's yeast strains secreted scu-PA(N302A) in a strongly growth-associated manner . Greater than 90% of the u-PA found in the culture broth was in the single-chain form . Scu-PA(N302A) was purified to homogeneity using two chromatography steps . The purified protein had a molecular weight of 47,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lacked any detectable N-linked glycosylation . The in vitro fibrinolytic properties of scu-PA(N302A) were found to be essentially equivalent to those of natural single-chain u-PA derived from the human kidney cell line TCL-598 . Since scu-PA(N302A) lacks the immunogenic N-linked carbohydrate pattern of yeast, it may be a useful therapeutic agent which can be produced economically by yeast fermentation.

J Mol Biol, 1990 Jan 5, 211(1), 235 - 48
Refined structure of yeast apo-enolase at 2.25 A resolution; Stec B et al.; The crystal structure of apo-enolase from baker's yeast (Saccharomyces cerevisiae) was established at 2.25 A resolution using a restrained least-squares refinement method . Based on 21,077 independent reflections of better than 8 A resolution, a final R-factor of 15.4% was obtained with a model obeying standard geometry within 0.017 A in bond length and 3.5 degrees in bond angles . The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.18 A . The refinement confirmed the heterodox, non-parallel character of the 8-fold beta alpha-barrel domain with beta beta alpha alpha(beta alpha)6 topology . The reported structure for which the data were collected at pH 5.0 represents an apo-form of the enzyme . Of the three carboxylic ligands that form the conformational metal ion binding site two, Glu295 and Asp320, are very close and presumably form a strong acidic type hydrogen bond with the proton partially replacing the electric charge of the physiological cofactor Mg2+ . The single sulfate ion found in the structure is in the active site cavity, co-ordinated to the side-chains of Lys345 and Arg374, and to the N atom of Ser375 . It is located about 7.4 A from the conformational metal ion binding site . It occupies the site in which the phosphate group of the substrate binds.

J Mol Biol, 1990 Jan 5, 211(1), 249 - 54
Three-dimensional structure of the complex of guanylate kinase from yeast with its substrate GMP; Stehle T et al.; The enzyme guanylate kinase was isolated from baker's yeast and crystallized as a complex with its substrate GMP . The crystal structure was solved by multiple isomorphous replacement, solvent-flattening, restrained least-squares refinement, and simulated annealing . The current R-factor is 28.9% at a resolution of 2.0 A . The model is given as a backbone tracing, the GMP binding site is shown in atomic detail . In its major domain (residues 1 to 32 and 82 to 186), the chain fold is closely similar to the adenylate kinases, while the minor domain (residues 33 to 81) differs grossly from the 3-helix fold of the adenylate kinases . Structural homology and mechanistical similarity allow us to assign the AMP site of the adenylate kinases on the basis of the GMP site.

Cytobios, 1990, 63(252), 15 - 22
Inhibitory effect of transfer RNA on protein kinases from baker's yeast and rat skeletal muscle; Kuo WN et al.; Sephadex G-200 gel filtration of DNA cellulose-treated crude extracts of rat skeletal muscle, revealed a broad peak-fraction of tRNA-inhibitory protein kinases (PK) coeluted endogenous substrates . In comparison, the elution profile of baker's yeast exhibited multiple peak-fractions of tRNA-inhibiting PK . Various tRNA all showed inhibition to PK . In the presence of regulatory subunit of cyclic AMP-dependent protein kinase, tRNA did not exert synergetic inhibition on PK . Moreover, the interaction of tRNA with active muscle PK fractions could not be monitored by the increment of absorbance at 340 nm . tRNA had no significant regulatory effect on the phosphorylation of actin and myosin.

Appl Biochem Biotechnol, 1990 Spring-Summer, 24-25, 565 - 78
In situ bubble fractionation strategies for separating individual proteins in a batch baker's yeast fermentation process; DeSouza AH et al.; Extracellular proteins produced by yeast have been observed to stratify in the extracellular fluid of a batch bioreactor, thus creating a vertical concentration gradient . We observed that, in the four different experiments conducted, each varied in their protein recovery characteristics . For example, sparging the system with gas accentuates the separation, though even in a nonsparged system, the in situ generation of minute carbon dioxide bubbles by yeast cells creates a protein gradient as the bubbles carry proteins upward . Based on these and other observations, we propose possible strategies for recovering the individual proteins from a system containing the four major proteins considered . A simple steady-state mathematical model, based on convective upward protein transport being balanced by downward protein diffusion, has been used to describe the behavior of each of these four extracellular proteins in the fermentation broth.

Biomed Biochim Acta, 1990, 49(6), 431 - 7
Phosphofructokinase from baker's yeast: studies on the initial kinetics and the fast reacting thiol groups of a proteolytically modified active enzyme form; Bar J et al.; The initial kinetics as well as the reactivity of the fast reacting thiol groups of a tetrameric form of phosphofructokinase from baker's yeast (called 12 S-enzyme), obtained by limited proteolysis in the presence of ATP were studied by the stopped-flow technique . Before attaining the steady state, the reaction shows a lag phase in the product formation, the duration of which decreases with increasing enzyme concentration . The lag phase disappears after preincubation of the enzyme with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate . The occurrence of an initial transient phase suggests that the enzyme converts from a state of low activity into a highly active one after starting the reaction . The modified enzyme was found to contain two fast reacting cysteinyl residues with respect to their reactivity towards 5,5'-dithiobis(2-nitrobenzoic acid) . Fructose 6-phosphate, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate, respectively, decrease the reactivity of this class of thiol groups but not the total number of titrable cysteins . This result supports the hypothesis of a conformational change as a consequence of the effector binding.

Arch Biochem Biophys, 1990 Jan, 276(1), 227 - 31
Studies of protein-protein interaction using countercurrent distribution in aqueous two-phase systems: partition behavior of five glycolytic enzymes from crude baker's yeast extract; Persson LO et al.; The partition behavior of five glycolytic enzymes, in extracts from baker's yeast (Saccharomyces cerevisiae), between two aqueous phases has been studied by countercurrent distribution . All enzymes showed distribution patterns which indicated homogeneity and a similar partition behavior . In purified form, three of the enzymes (glyceraldehyde-phosphate dehydrogenase, 3-phosphoglycerate kinase, and enolase) showed the same partition behavior as in the extracts . Pure 6-phosphofructokinase, on the other hand, changed its partition distinctively relative to what was found in the extracts . These results indicate interactions between this enzyme and macromolecular compounds in the extracts and support a model suggested by Kurganov et al . (1985, J . Theor . Biol . 116, 509-526) describing a "glycolytic particle."

Bioseparation, 1990, 1(3-4), 235 - 54
Affinity partitioning and extraction of proteins; Kopperschlager G et al.; Affinity partitioning of enzymes and plasma proteins in aqueous two-phase systems has been reviewed . Besides basic theoretical considerations of the principle of affinity partitioning the chemistry of coupling ligands to the polymers, the nature and properties of selected biomimetic ligands like dye-ligands, immunoligands, metal chelate ligands and hydrophobic ligands are reported . The usefulness of affinity partitioning for studying the affinity of ligands and their specificity to proteins has been demonstrated by selected examples . The method proved also applicable to study the structural dynamics of proteins as exemplified with phosphofructokinase from baker's yeast and human alpha-2-macroglobulin . The current knowledge of metal chelate affinity partitioning is presented as well as the applicability of affinity partitioning for the purification of enzymes.

Bioprocess Technol, 1990, 9, 67 - 94
Protein secretion systems in microbial and mammalian cells; Moir DT et al.; Secretion is an attractive production mode for proteins that require posttranslational modifications carried out in the secretory pathway . For example, amino acid chains fold properly, disulfide bonds form correctly, and glycosylation occurs accurately as the protein is secreted . In addition, recovery of secreted proteins is simplified by the fact that cells need not be broken and the product may be a major species present in a minimal synthetic culture medium . The current state of the art permits the production and secretion of proteins by heterologous cells . While future efforts will be required to improve the efficiency of secretion, current methods yield commercially interesting levels of secretion from E . coli, B . subtilis, S . cerevisiae, Aspergilli, and many mammalian cell types . Each of these systems offers certain advantages, and the choice of system depends on the specific protein to be secreted . High-value human therapeutic products such as plasminogen activators are reasonable candidates for secretion from mammalian cell lines, while industrial proteins such as calf prochymosin require a more economical host, such as baker's yeast or Aspergillus . The wide variety of posttranslational modifications observed in nature may prevent any single secretion system from dominating the field for many years to come.

J Biol Chem, 1989 Dec 25, 264(36), 21619 - 20
Preliminary crystallographic data for transketolase from yeast; Schneider G et al.; Crystals of the vitamin B1-dependent enzyme transketolase from baker's yeast have been grown from the apo- and the holoform of the enzyme, using PEG as precipitant . The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell constants a = 76.3 A, b = 114.2 A, and c = 163.5 A . The crystals are stable in the x-ray beam and diffract to at least 2.2 A on a conventional x-ray source . The enzyme is a dimer of identical subunits, and a Vm value of 2.2 A/dalton indicates that the asymmetric unit contains a dimer . Rotation function calculations using native data (10-5 A) revealed a local 2-fold rotation axis with phi = 0 degree and omega = 20 degrees.

Eur J Biochem, 1989 Dec 22, 186(3), 535 - 41
Arginyl-tRNA synthetase from yeast . Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2); Freist W et al.; For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values . The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations . The other noncognate amino acids exhibit D values between 10,000 and 60,000 . In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600 . From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role . Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors . According to a two-step binding process, two factors (I1 and I2) were determined . They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain . A hypothetical 'stopper' model of the amino acid recognition site is discussed.

Proc Natl Acad Sci U S A, 1989 Nov, 86(22), 8847 - 51
In vivo rearrangement of mitochondrial DNA in Saccharomyces cerevisiae; Clark-Walker GD; A revertant (SPR1) from a high-frequency petite strain of Saccharomyces cerevisiae has been shown by mapping and sequence analysis to have a rearranged mitochondrial genome . In vivo rearrangement has occurred through a subgenomic-recombination pathway involving the initial formation of subgenomic molecules in nascent petite mutants, recombination between these molecules to form an intermediate with direct repeats, and subsequent excision of the resident or symposed duplication to yield a molecule with three novel junctions and a changed gene order . Sequencing of the novel junctions shows that intramolecular recombination in each case occurs by means of G + C-rich short direct repeats of 40-51 base pairs . Mapping and sequence analysis also reveal that the SPR1 mitochondrial genome lacks three sectors of the wild-type molecule of 4.4, 1.7, and 0.5 kilobases . Each of these sectors occurs in nontemplate, base-biased DNA, that is over 90% A + T . Absence of these sectors together with a rearranged gene order does not appear to affect the phenotype of SPR1, as colony morphology and growth rate on a number of different substrates are not detectably different from the wild type . Lack of phenotypic change suggests that mitochondrial gene expression has not been noticeably disrupted in SPR1 despite deletion of the consensus nonomer promoter upstream from the glutamic acid tRNA gene . Dispensability of DNA sectors and the presence of recombinogenic short, direct repeats are mandatory features of the subgenomic-recombination pathway for creating rearrangements in baker's yeast mtDNA . It is proposed that, in other organisms, organelle genomes containing these elements may undergo rearrangement by the same steps.

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8266 - 70
Higher-order structure of Saccharomyces cerevisiae chromatin; Lowary PT et al.; We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding . This has required the use of yeast strains that are free of the ubiquitous yeast "killer" virus . Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells . These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1 . Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure.

Biochem J, 1989 Sep 15, 262(3), 897 - 908
Purification and characterization of recombinant human interleukin 4 . Biological activities, receptor binding and the generation of monoclonal antibodies; Solari R et al.; A synthetic gene coding for human interleukin 4 (IL-4) was cloned and expressed in Saccharomyces cerevisiae (baker's yeast) as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence, resulting in secretion of mature IL-4 into the culture medium (0.6-0.8 micrograms/ml) . A protocol was developed for purification of this protein . Crude cell-free conditioned medium was passed over a concanavalin A-Sepharose affinity column; bound proteins were eluted and further purified by S-Sepharose Fast Flow cation exchange and C18 reverse-phase h.p.l.c . Highly purified IL-4 was obtained by this method (0.3-0.4 mg per litre of culture) with a recovery of 51% . Thermospray liquid chromatography-mass spectrometry showed the C-terminal N-glycosylation site to be largely unmodified, and also showed that the N-terminus of the purified recombinant IL-4 (rIL-4) was authentic . Thiol titration revealed no free cysteine residues, implying that there are three disulphide groups, the positions of which remain to be determined . We have characterized the biological activities of the purified rIL-4 . This material is active in B-cell co-stimulator assays, T-cell proliferation assays and in the induction of cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells . Half-maximal biological activity of the rIL-4 was achieved at a concentration of 120 pM . We have radioiodinated rIL-4 without loss of biological activity and performed equilibrium binding studies on Raji cells, a human B-cell line . The 125I-rIL-4 bound specifically to a single class of binding studies on Raji cells, a human B-cell line . The 125I-rIL-4 bound specifically to a single class of binding site with high affinity (Kd = 100 pM) and revealed 1100 receptors per cell . Receptor-ligand cross-linking studies demonstrated a single cell-surface receptor with an apparent molecular mass of 124 kDa . Two monoclonal antibodies have been raised to the human rIL-4, one of which blocks both the biological activity of rIL-4 and binding to its receptor.

Biochim Biophys Acta, 1989 Aug 31, 997(3), 159 - 66
An examination of the role of arginine residues in the functioning of D-glyceraldehyde-3-phosphate dehydrogenase; Asryants RA et al.; Chemical modification of one arginine residue per subunit of tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) molecule results in a 85-95% loss of its activity (Nagradova and Asryants (1975) Biochim . Biophys . Acta 386, 365-368; Nagradova, N.K., Asryants, R.A., Benkevich, N.V . and Safronova, M.I . (1976) FEBS Lett . 69, 246-248) . Transient kinetic experiments performed in the present work with modified rabbit muscle and Baker's yeast enzymes showed that the first-order rate constant of acyl-enzyme.NADH formation was diminished 30-fold with the rabbit muscle enzyme and 60-fold with the Baker's yeast enzyme . Modification of arginine residues was shown also to affect the second step of the catalytic reaction, the phosphorolysis of the acyl-enzyme (the second-order rate constant of phosphorolysis decreased 9-fold in the case of the rabbit muscle enzyme and 40-fold in the case of the Baker's yeast enzyme) . The native and modified enzymes exhibited similar inhibitory constant values with respect to NADH, suggesting no contribution of arginine residues to the acyl-enzyme.NADH complex destabilization . By and large, the experimental data are consistent with the hypothetical scheme proposed on the basis of X-ray crystallography studies to describe a participation of Arg-231 in the catalytic mechanism of D-glyceraldehyde-3-phosphate dehydrogenase (Grau (1982) in the Pyridine Nucleotide Coenzymes, p . 135-187).

Pharmazie, 1989 Aug, 44(8), 558 - 60
Birch sap and birch leaves extract: screening for antimicrobial, phagocytosis-influencing, antiphlogistic and antipyretic activity; Klinger W et al.; Birch sap and birch leaves extract have been screened for antimicrobial, phagocytosis-influencing, anti-inflammatory and antipyretic activity . No antimicrobial effects could be detected in the agar-diffusion test with staphylococcus as test strain, whereas birch sap exhibited some inhibitory effect on phagocytosis, which exceeded that of the citric acid added to the birch sap as preservative . In rats, only the high doses of 1 and 2 ml/100 g b.m . birch sap had a weak and short-lasting anti-inflammatory activity against the Carrageenin edema, whereas birch leaves extract proved to be ineffective . Fever induced by baker's yeast in rats was inhibited by birch leaves extract in the high dose of 4 ml/100 g b.m . significantly, but not by birch sap, and only for a short period . Acetylsalicyclic acid had a much higher anti-inflammatory and antipyretic activity . Altogether despite of detectable anti-inflammatory, antipyretic and phagocytosis-inhibiting effects of these birch products no therapeutic activity of importance compared with classical and modern antipyretics-analgetics can be demonstrated.

Curr Genet, 1989 Jul, 16(1), 13 - 20
Biosynthesis of the peroxisomal dihydroxyacetone synthase from Hansenula polymorpha in Saccharomyces cerevisiae induces growth but not proliferation of peroxisomes; Godecke A et al.; The DAS gene of Hansenula polymorpha was expressed in Saccharomyces cerevisiae under the control of different promoters . The heterologously synthesized dihydroxyacetone synthase (DHAS), a peroxisomal enzyme in H . polymorpha, shows enzymatic activity in baker's yeast . The enzyme was imported into the peroxisomes of S . cerevisiae not only under the appropriate physiological conditions for peroxisome proliferation (oleic acid media), but also in glucose-grown cells where it induced the enlargement of the few peroxisomes present . This growth process was not accompanied by an increase in the number of microbodies, which suggests a separate control mechanism for peroxisomal proliferation.

Eur J Biochem, 1989 Jun 15, 182(2), 451 - 6
Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase; Smirnova IN et al.; Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis . In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme . A series of absorbance versus Pi concentration curves in the presence of 0.5-20 mM free Mg2+ were obtained at pH 7.2 and computer-fitted to 19 models . The dissociation constant of magnesium phosphate (8.5 +/- 0.4 mM) used in this analysis was measured with a Mg2+-sensitive electrode . The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules . The first route predominates at physiological concentrations of Mg2+ . The Pi-inhibition pattern of pyrophosphate hydrolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pi-binding site . The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.

Arch Biochem Biophys, 1989 May 15, 271(1), 240 - 5
NAD-linked, GSH- and factor-independent aldehyde dehydrogenase of the methylotrophic bacterium, Hyphomicrobium X; Poels PA et al.; Cell-free extracts of Hyphomicrobium X showed NAD-dependent aldehyde dehydrogenase activity, provided that NAD addition preceded that of aldehyde . Activity was lost rather rapidly, especially during purification attempts, but this could be partially masked by including a time-dependent restoration step with thiol compounds in the protocol . The nature of the assay buffer appeared to be critical and stimulation occurred on incorporation of K+ ions in the mixture . An even higher specific activity could be achieved by 1,4-dithiothreitol (DTT) treatment of the preparation, followed by removal of DTT, and assaying in the absence of thiol compounds under anaerobic conditions . Exposure of such a preparation to O2 led to a significant decrease in activity within a couple of hours . Immediate inactivation occurred on addition of H2O2, but this could be prevented completely by prior addition of NAD . Since GSH does not participate in the reaction and no stimulating factor was detected, the role of thiol compounds is most probably confined to restoration or prevention of damage to an O2-sensitive, necessary thiol group . Since the same features were found for cell-free extract as for the partially purified enzyme, only one enzyme type seems to be present . Although the enzyme is a general aldehyde dehydrogenase, the kinetic parameters and the specific activity of the cell-free extract for formaldehyde indicate that it may play a role in formaldehyde dissimilation by Hyphomicrobium X . The NAD-linked, GSH- and factor-independent aldehyde dehydrogenase described here appears to be different in several respects from the formaldehyde dehydrogenase of Pseudomonas putida (EC 1.2.1.46) (despite showing similar behavior toward coenzymes and factors) but resembles the aldehyde dehydrogenase from baker's yeast (EC 1.2.1.5).

Mol Gen Genet, 1989 Mar, 216(1), 149 - 55
Control of formation of active soluble or inactive insoluble baker's yeast alpha-glucosidase PI in Escherichia coli by induction and growth conditions; Kopetzki E et al.; Using standard growth conditions (LB medium, 37 degrees C, induction with 5 mM IPTG) yeast alpha-glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated alpha-glucosidase granules in Escherichia coli . Under these conditions active soluble alpha-glucosidase amounts to less than 1% of the heterologously produced protein . However, the amount of soluble active alpha-glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced . This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactose-permease deficient host strain containing the lacIq repressor gene on an R-plasmid . The formation of active soluble alpha-glucosidase was almost 100% when E . coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e . at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.

Biochem J, 1989 Mar 1, 258(2), 473 - 8
pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase; Ascenzi P et al.; The spectral (e.p.r . and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined . At neutral pH the e.p.r . and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively . By lowering pH, the e.p.r . and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms . By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond . However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant.

Biochem J, 1989 Feb 15, 258(1), 109 - 13
Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues; Loveless RW et al.; Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides . The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II {Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc} respectively.

Can J Microbiol, 1989 Feb, 35(2), 295 - 303
Purification and properties of three endopeptidases from baker's yeast; Nowak J et al.; Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae . Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained . Both forms were inhibited by pepstatin and other acid proteinase inhibitors . The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0 . The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea . Urea also stimulated the enzyme activity by 30-50% . As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type . A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases . The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months . The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB . Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin . On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol . The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1989 Jan 2, 242(2), 351 - 6
Identification of the major tRNA(Phe) binding domain in the tetrameric structure of cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast; Fasiolo F et al.; Native cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast is a tetramer of the alpha 2 beta 2 type . On mild tryptic cleavage it gives rise to a modified alpha 2 beta 2 form that has lost the tRNA(Phe) binding capacity but is still able to activate phenylalanine . In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated beta subunit . Each purified peptide was unambiguously assigned to a unique stretch of the beta subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing . Together with earlier results from affinity labelling studies the present data show that the Lys 172-Ile 173 bond is the unique target of trypsin under mild conditions and that the N-terminal domain of each beta subunit (residues 1-172) contains the major tRNA(Phe) binding sites.

Eur J Biochem, 1989 Jan 2, 178(3), 595 - 602
Isoleucyl-tRNA synthetase from baker's yeast . Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA; role of terminal hydroxyl groups aminoacylation of tRNA(Ile)-C-C-A; Freist W et al.; Specificity with regard to amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA by isoleucyl-tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values . The lowest values are observed for Cys, Val, His, and Trp (D2 = 180-1700), indicating that at same amino acid concentrations isoleucine is 180-1700 times more attached to tRNA(Ile)-C-C-3'dA . The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2 = 10,000-30,000) . D2 values of the other amino acids are in the range of 2000-10,000 . Recognition of most amino acids is achieved in a four-step process . Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof-reading steps occur on the pre- and the post-transfer stage . For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post-transfer proof-reading is negligible . As a special case in discrimination of valine, one initial discrimination step and the post-transfer proof-reading step are lacking . The role of the terminal hydroxyl groups of the tRNA for post-transfer proof-reading is assigned to a simple neighbouring group effect . No preference for the 2' or 3' position in proof-reading can be postulated.

Cytobios, 1989, 59(238-239), 177 - 83
Interaction between cortisol and microbial proteases; Kuo WN et al.; Binding (or interaction) of cortisol with microbial molecule(s) was observed by employing Bio-Gel HTP affinity chromatography and subsequently by fluorescence spectrophotometry . Molecule(s) in the crude extract of baker's yeast and in other microbial proteases exhibited varied degrees of cortisol-binding . Bacterial protease (type IX) had highest, while the type XXVI enzyme had the lowest, binding capacity . In addition, these two proteases exhibited a distinct difference in the alterations of ultraviolet spectra due to interaction with cortisol . Using casein as a substrate, cortisol, CTP, trypsin inhibitor or leupeptin appreciably inhibited type IX protease at low concentrations of Ca2+ . However, thyroxine had no effect on this protease.

Soz Praventivmed, 1989, 34(2), 53 - 7
{Microorganisms in our food: yesterday, today and tomorrow}; Niederberger P; The development of fermented foodstuffs can be considered as one of the greatest achievements of human civilization and goes back thousands of years . It arose from the necessity to conserve foods, to make them more digestible and also more enjoyable . During the centuries man learned by trial and error to conduct the fermentation processes by changing physical and chemical parameters in the basic food ingredients . It took much longer, however, to discover the cause of fermentation processes: only towards the end of the 19th century it was demonstrated, for example, that baker's yeast is the cause of alcoholic fermentation . Through the development of the technique of "pure microbial culture", developed again with yeast, the basis for a controlled use of microbes in food industry was established . The movement from artisan to industrial scale fermentations made necessary a systematic strain development by the "classical" techniques of "mutation, selection and recombination" . The modern techniques of genetic engineering have hardly been applied to food microorganisms so far . These methods, which usually have been developed with laboratory strains, would give us the possibility to change or disrupt genes very specifically and even to introduce new genes into established food microorganisms . Some possible applications with the baker's yeast are discussed in this article.

Yeast, 1989 Jan-Feb, 5(1), 11 - 24
Cloning and characterization of baker's yeast alpha-glucosidase: over-expression in a yeast strain devoid of vacuolar proteinases; Kopetzki E et al.; Two alpha-glucosidase (maltase) genes, designated GLUCPI and GLUCPII, have been cloned from an industrial strain of baker's yeast (Saccharomyces cerevisiae) by complementation of a maltase-negative mutant strain . The different genes were identified according to their alternatively expressed isoenzymes PI and PII in transformants after isoelectric focusing and activity staining in separated cell lysates . The gene encoding alpha-glucosidase PI (GLUCPI), which was not present in laboratory strains of S . carlsbergensis with a defined MAL1, 2, 3, 4 or 6 locus, was sequenced and compared with the recently published MAL6S gene . This comparison revealed single amino acid deviations at three positions in the predicted polypeptide sequence . In addition, the divergent promoter region of GLUCPI differed from MAL6S by a triple repeated 147-bp DNA segment . Maltose induction and glucose repression of alpha-glucosidase PI were not affected by the deletion of the repeated DNA segment . However, the absolute expression of alpha-glucosidase PI increased two- to four-fold . In addition, a two-fold increase in the maltase synthesis occurred when the cloned positive regulator gene MAL2-8ep was on the same plasmid . Furthermore, stability of the alpha-glucosidase in cultures in the stationary growth phase was greatly enhanced using a host strain lacking the proteinases A and B and the carboxypeptidases Y and S . Promoter trimming, MAL2-8cp stimulation and the use of a host strain deficient in four vacuolar proteinases resulted in alpha-glucosidase PI expression of about 13% of the soluble protein.

Prog Clin Biol Res, 1989, 318, 81 - 9
DNA sequence analysis of newly formed telomeres in yeast; Wang SS et al.; A plasmid can be maintained in linear form in baker's yeast if it bears telomeric sequences at each end . Linear plasmids bearing cloned telomeric C4A4 repeats at one end (test end) and a natural DNA terminus with approximately 300 bps of C4A2 repeats at the other or control end were introduced by transformation into yeast . Test-end termini of 28 to 112 bps supported telomere formation . During telomere formation, C4A2 repeats were often transferred to test-end termini . To determine in greater detail the fate of test-end sequences on these plasmids after propagation in yeast, test-end telomeres were subcloned into E . coli and sequenced . DNA sequencing established a number of points about the molecular events involved in telomere formation in yeast . The results suggest that there are at least two mechanisms for telomere formation in yeast . One is mediated by a recombination event that requires neither a long stretch of homology nor the RAD52 gene product . The other mechanism is by addition of C1-3A repeats to the termini of linear DNA molecules . The telomeric sequence required to support C1-3A addition need not be at the very end of a molecule for telomere formation.

Cell Signal, 1989, 1(1), 1 - 7
Transmembrane signalling in Saccharomyces cerevisiae; Engelberg D et al.; Baker's yeast, a unicellular eukaryote, has been a model organism for biochemists, geneticists and most recently for molecular biologists . Pioneering biochemical studies were conducted on yeast, such as the study of glucose fermentation and amino acid metabolism . The powerful tools of yeast genetics have allowed a comprehensive study of important issues such as the cell cycle and meiosis . In recent years, it has been established that Saccharomyces cerevisiae, the most extensively characterized of the yeasts, shares key molecules and biochemical pathways with higher eukaryotes . For example, actin, tubulin, ubiquitin, calmodulin, GTP regulatory proteins, different protein kinases including protein tyrosine kinases, were all found to play central roles in yeast . Furthermore, structurally homologous proteins, as well as transcription regulating elements, of yeast and higher eukaryotes, including mammals, were shown to be structurally and functionally interchangeable . It has also been found that yeast can express human genes . Technically, yeasts are simple to handle, inexpensive to grow, complete a cell cycle within 90 min, and therefore can yield relatively quick results . These qualities are useful in biotechnological applications . Saccharomyces cerevisiae, can be genetically manipulated fairly easily, and has been tinkered with more than any other system . A cloned, in vitro mutated gene, can be transformed into wild type yeast and by homologous recombination, can replace the native gene and generate the desired mutant . Such manipulations, not possible yet in other eukaryotic cells, allow the precise definition of the role played by different genes and their domains . These unique features of Saccharomyces cerevisiae, together with rapidly evolving techniques of molecular biology, have made it a successful model organism for the study of numerous questions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomed Biochim Acta, 1989, 48(7), 387 - 92
Phosphofructokinase from baker's yeast: kinetic properties of a proteolytically modified enzyme; Bar J et al.; A tetrameric enzyme form of phosphofructokinase from yeast (called 12 S-enzyme), formed by limited proteolysis of the octameric enzyme in the presence of ATP and by subsequent dissociation in two half-molecules shows sigmoidal kinetics with respect to fructose 6-phosphate and inhibition by ATP . Similar to the native phosphofructokinase, the modified enzyme is also efficiently activated by AMP and fructose 2,6-bisphosphate . Both activators increase the affinity for the substrate fructose 6-phosphate and the respective maximum activity . In contrast to the native phosphofructokinase, however, both AMP and fructose 2,6-bisphosphate change the sigmoidal fructose 6-phosphate velocity curve into a hyperbolic one . AMP and fructose 2,6-bisphosphate decrease the ATP inhibition, probably by modulating the affinity of the allosteric sites to ATP.

Int J Immunopharmacol, 1989, 11(2), 191 - 5
Antitumor effect of a baker's yeast mannan-mitomycin C conjugate against mouse hepatoma, MH134, in vivo and in vitro; Aizawa K et al.; A conjugate of mitomycin C and the mannan of bakers' yeast (Saccharomyces cerevisiae wild type strain) was synthesized . Assay of its growth inhibitory effect on MH134 hepatoma solid tumor implanted in C3H/He slc mice showed that this conjugate exhibited a higher growth-inhibition ratio than those of free mitomycin C and the same bakers' yeast mannan in the corresponding effective doses . This conjugate was also found to kill the same hepatoma cells in vitro . Because a dextran-mitomycin C conjugate did not manifest any antitumor effect against this tumor, it was postulated that interaction of the mannan moiety in the mannan-mitomycin C conjugate with the mannose receptor located on the cell surface of immunocytes and/or with hepatoma cells participated in the common initiating reaction of in vitro and in vivo antitumor effects.

Biochim Biophys Acta, 1988 Nov 2, 957(1), 60 - 70
D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms; Douzhenkova IV et al.; Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit . Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer . A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms . In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer . The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions . Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+ . At coenzyme concentrations below half-saturating a monomer is more active than a tetramer . This difference disappears at saturating concentrations of NAD+ . Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis . The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis . This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated . Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1988 Nov 1, 177(2), 425 - 33
Tyrosyl-tRNA synthetase from baker's yeast . Order of substrate addition, discrimination of 20 amino acids in aminoacylation of tRNATyr-C-C-A and tRNATyr-C-C-A(3'NH2); Freist W et al.; The order of substrate addition to tyrosyl-tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead-end inhibitors . The kinetic patterns are consistent with a random bi-uni uni-bi ping-pong mechanism . Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate . Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from kcat and Km values obtained in aminoacylation of tRNATyr-C-C-A . The lowest values are observed for Cys, Phe, Trp (D = 28,000-40,000), showing that, at the same amino acid concentrations, tyrosine is 28,000-40,000 times more often attached to tRNATyr-C-C-A than the noncognate amino acids . With Gly, Ala and Ser no misacylation could be detected (D greater than 500,000); D values of the other amino acids are in the range of 100,000-500,000 . Lower specificity is observed in aminoacylation of the modified substrate tRNATyr-C-C-A(3'NH2) (D1 = 500-55,000) . From kinetic constants and AMP-formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATyr-C-C-A hydrolytic proof-reading factors could be calculated for a pretransfer (II 1) and a post-transfer (II 2) proof-reading step . The observed values of II 1 = 12-280 show that pretransfer proof-reading is the main correction step whereas post-transfer proof-reading is marginal for most amino acids (II 2 = 1-2) . Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof-reading factors . Assuming a two-step binding process, two factors (I1 and I2) are determined which can be related to hydrophobic interaction forces . The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group . A hypothetical model of the amino acid binding site is discussed and compared with results of X-ray analysis of the enzyme from Bacillus stearothermophilus.

Eur J Pharmacol, 1988 Oct 18, 155(3), 301 - 3
Exocytosis as a possible mechanism for the secretion of coproporphyrin from a chloroquine-treated yeast; Kotal P et al.; Chloroquine, which is known to be concentrated in intracellular acid vesicles, stimulates the release of porphyrins from yeast . Experiments with normal baker's yeast Saccharomyces cerevisiae and the sec-1 mutant, with suppressed exocytosis, showed that the release of porphyrins was stimulated more in normal yeast than it was in the mutant . It is suggested that chloroquine stimulates the exocytosis of porphyrins.

Biochemistry, 1988 Sep 20, 27(19), 7365 - 71
Rat kidney L-2-hydroxyacid oxidase . Structural and mechanistic comparison with flavocytochrome b2 from baker's yeast; Urban P et al.; Hydroxyacid oxidase from rat kidney is an FMN-dependent enzyme that catalyzes the oxidation of L-alpha-hydroxy acids as well as, more slowly, that of L-alpha-amino acids . We report here a modified purification method for the enzyme, which is found to possess one cofactor per subunit of Mr 39,000 . Determination of its N-terminal sequence suggests the protein is homologous to spinach glycolate oxidase and baker's yeast lactate dehydrogenase . In the presence of a hydroxy acid and of bromopyruvate, under anaerobic conditions, the enzyme is found to catalyze both transhydrogenation and reductive bromide ion elimination . It had previously been observed that hydroxyacid oxidase could not catalyze chloride elimination from chlorolactate in the presence of oxygen {Cromartie, T.H., & Walsh, C.T . (1975) Biochemistry 14, 3482-3490} . The behavior of this enzyme toward halogeno substrates is therefore similar to that of baker's yeast L-lactate dehydrogenase and in part different from that of Mycobacterium smegmatis lactate oxidase and porcine kidney D-amino-acid oxidase . These findings can be rationalized on the basis of a common mechanism for all these enzymes, implying formation of a carbanion as a first step, with different rate-limiting steps in the overall reaction.

Biochem Int, 1988 Sep, 17(3), 517 - 21
Inhibition of transketolase by N-acetylimidazole; Kuimov AN et al.; Transketolase from baker's yeast is rapidly inactivated in the presence of N-acetylimidazole . According to kinetic data, acetylation of one amino acid residue of the protein per active site is sufficient for TK* inactivation . The holoenzyme is inhibited more slowly than is apotransketolase . The presence of a tyrosine residue in the enzyme's active site, essential for activity, is suggested.

Appl Biochem Biotechnol, 1988 Aug, 18, 3 - 17
Semisolid state fermentation of baker's yeast in an air-fluidized bed fermentor; Hong K et al.; In an attempt to grow microorganisms other than fungi using a solid-state fermentation process, a model system of Baker's yeast (Saccharomyces cerevisiae) was cultured in an air-fluidized bed fermentor . A semisolid potato mixture (pretreated with alpha-amylase) was used for the substrate in this highly aerated system . The growth of Baker's yeast in this air-fluidized bed process was easily controllable and very reproducible . Once feasible moisture levels and air flow rates were determined, the independent variables studied were the amount of the enzyme used for digesting the potato starch, the size of the yeast inoculum, and the concentration of the added defined medium.

Biochem Biophys Res Commun, 1988 Jul 15, 154(1), 358 - 64
Partial amino acid sequence of rat topoisomerase I: comparison with the predicted sequences for the human and yeast enzymes; Durban E et al.; The partial amino acid sequence of rat topoisomerase I was determined by gas-phase microsequencing . Seven tryptic peptides closely matched the sequences deduced from human topoisomerase I cDNA (94.5% homology) . Similarity to sequences deduced from baker's yeast and fission yeast genomic DNA were restricted to conserved domains which may represent important sites of interaction with DNA or with other proteins.

Biofactors, 1988 Jul, 1(2), 187 - 92
Physical and kinetic properties of a pyridoxal reductase purified from bakers' yeast; Guirard BM et al.; Pyridoxine dehydrogenase (1.1.1.65) (pyridoxal reductase), purified to homogeneity from baker's yeast, is a monomer of Mr approximately 33,000 . It catalyzes the reversible oxidation of pyridoxine by NADP to yield pyridoxal and NADPH; equilibrium lies far in the direction of pyridoxine formation (Keq approximately 1.4 X 10(11) l/mol at 25 degrees C) . Reduction of pyridoxal occurs most rapidly at pH 6.0-7.0; oxidation of pyridoxine is optimal at pH 8.6 . NAD and NADH do not replace NADP and NADPH as substrates; pyridoxine, pyridoxal and pyridoxal 5'-phosphate are the only naturally occurring cosubstrates found . Several other aromatic aldehydes also are reduced, but substrate specificity and other properties of the enzyme distinguish it clearly from other alcohol dehydrogenases or aldehyde reductases . Between pH 6.3 and 7.1 (the intracellular pH of yeast), V/Km with pyridoxal and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADP as substrates . These and other considerations strongly indicate that the dehydrogenase functions in vivo to reduce pyridoxal to pyridoxine, which is the preferred substrate for pyridoxal (pyridoxine) kinase in yeast.

Biochem J, 1988 Jul 1, 253(1), 109 - 16
The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker's yeast (Saccharomyces cerevisiae); Kurlandzka A et al.; Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis . The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV . Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100% . This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo . The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps . The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin . In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation . The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.

Biol Chem Hoppe Seyler, 1988 Jun, 369(6), 417 - 24
Studies on the citryl-CoA-dependent inhibition of citrate-synthase with source variants from baker's yeast, Escherichia coli and Sulfolobus solfataricus; Lohlein-Werhahn G et al.; 1) Citrate synthase from pig heart has previously been shown to display complex kinetic characteristics in the reactions with citryl-CoA, resulting in inhibition . The synthase from another eukaryotic source, baker's yeast, yields the same complex kinetics . 2) Synthases from a Gram-negative prokaryote, E . coli, and from an archaebacterium, S . solfataricus, catalyse the reactions of citryl-CoA in kinetics of the Michaelis-Menten type . A comparison of the rates of citryl-CoA hydrolysis (V') and physiological reaction (V), determined with these enzymes, corresponds to ratios of V'/V approximately 1 and approximately 2, respectively . Thus, and for the first time, there is no reason left to doubt the intermediate formation of citryl-CoA in the physiological reaction . 3) The complex kinetics indicated under 1) are related to efficient formation of citrate from citryl-CoA-derived acetyl-CoA and oxaloacetate in the presence of NADH and malate dehydrogenase . These conditions are not met by the enzymes from E . coli, S . solfataricus and by proteolytically nicked synthase species from pig heart . All these enzyme variants have low affinities to either one or both of the physiological substrates . Consistent with earlier ideas, the results indicate that the inhibition mechanism is related to high affinities of the enzyme for both acetyl-CoA and oxaloacetate.

Ecotoxicol Environ Saf, 1988 Jun, 15(3), 253 - 9
Terrestrial model food chain and environmental chemicals . I . Transfer of sodium {14C}pentachlorophenate between springtails and carabids; Gruttke H et al.; A model soil food chain of a ruderal ecosystem has been constructed in order to study the uptake, transfer, and accumulation of {14C}pentachlorophenate (PCP-Na) . The model was based on three food levels, viz . baker's yeast, collembola, and carabid beetles, and the contaminant chemical introduced was via initial food . Continuous exposure of the organisms to the test chemical resulted in a significant uptake and transfer of radiocarbon into the food chain elements . Bioaccumulation of radiocarbon in the body tissues of the organisms was low, as large amounts taken up were quickly eliminated through the excrements . The radiocarbon level of prey animals was about 100 times higher than that of their predators, but there was only small difference in concentration between collembolas and yeast . This was probably because of a faster excretion of the chemical by the beetles than by the collembolas . During the test period no conversion of {14C}PCP-Na took place in the yeast, but the collembolas and beetles metabolized 50 and 59%, respectively . Criteria are proposed for successful implementation of food chain models.

Biochem Biophys Res Commun, 1988 May 16, 152(3), 981 - 6
Restoration of high-potential cytochrome b-564 by integration of baker's yeast complex III into liposomes; Hervas M et al.; Cytochrome b-564 in isolated complex III from baker's yeast mitochondria exhibits the midpoint redox potential proper to the low-potential couple (+60 mV, pH 7.2) . Incorporation of the complex into liposomes promotes total conversion to the high-potential couple (+170 mV, pH 7.2) . The reconstituted system shows electrogenic proton translocation, which is inhibited by the uncoupler CCCP . Deenergizing treatments result, moreover, in reversal of the redox potential change . These results support our previous proposal that cytochrome b-564 acts as a transducer of redox energy into acid-base energy in the complex III region of the respiratory chain.

FEBS Lett, 1988 May 9, 232(1), 121 - 4
3':5'-cyclic GMP in the yeast Saccharomyces cerevisiae at different metabolic conditions; Eckstein H; cGMP is characterized as undetectable in yeast {(1986) Yeast Cell Biology, UCLA Symp . Mol . Cell Biol . (Hicks, J . ed.) p . 495}, though in many organisms it contributes specifically to the regulation of metabolism . Here, we detected cGMP, using radioactive labeling and RIA techniques, after extraction of the cells with 1 mol/1 HClO4 at 37 degrees C . The cGMP 0.015-fold cAMP, about 3-times higher with exponentially growing cells than with pressed baker's yeast, and depends on glucose and O2 supply . The PDE inhibitors DMX and IBMX induce in growing cells an additional increase of the cGMP level, without similar effects on cAMP.

Z Naturforsch {C}, 1988 May-Jun, 43(5-6), 386 - 96
Evidence for cyclic GMP in the yeast Saccharomyces cerevisiae, and studies on its possible role in growth; Eckstein H; The yeast Saccharomyces cerevisiae is shown to be equipped with cyclic GMP, the level of which ranges from 6 pmol/10(9) cells with pressed baker's yeast to 21 pmol/10(9) cells with exponentially growing cells . In extracts from synchronized growing yeast, cyclic GMP increases stepwise, being doubled at the time of each mitosis . Theophylline and 3-isobutyl-1-methylxanthine induce a rapid increase of cyclic GMP, followed by a premature formation of the septal cell wall between mother cell and bud . The effects of 3-isobutyl-1-methylxanthine are reversible . Dibutyryl-cyclic GMP, and, after a pronounced lag, also dibutyryl-cyclic AMP, induce a premature cell division, too . Cholera toxin induces premature cell divisions without a preceding increase in cyclic GMP . Neither theophylline nor 3-isobutyl-1-methylxanthine, cholera toxin or one of the dibutyryl-cyclic nucleotides modify the growth rate of the culture . None of the agents has significant effects on the level of cyclic AMP . The results suggest that cyclic GMP possibly controls an early step of mitosis, whereas ADP-ribosylation might govern a subsequent event.

Biochim Biophys Acta, 1988 Apr 15, 959(3), 214 - 9
Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of {11C}acetylcoenzyme A; Mannens G et al.; The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 mumol {11C}acetylcoenzyme A . (CoA-{11C}Ac) . A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed . Several silanes, spacer arms and terminal reactive groups were tested . The immobilized enzyme was subjected to storage stability tests . From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass . The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads . The latter were packed in a glass column . The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes . This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.

Eur J Biochem, 1988 Apr 5, 173(1), 155 - 62
Inactivation of flavocytochrome b2 with fluoropyruvate . Reaction at the active-site histidine; Urban P et al.; Fluoropyruvate inactivated oxidized flavocytochrome b2 (baker's yeast L-lactate dehydrogenase) in a biphasic process yielding convex semilog plots of residual activity versus time . At each reagent concentration, rate constants k1 and k2 for the two phases could be calculated by simulation studies using one of the schemes proposed by Ray and Koshland {J . Biol . Chem . (1961) 236, 1973-1979}: E----E1 (fully active)----E2 (inactive) . When plotted as a function of reagent concentration, the values of k2, but not those of k1, showed a saturation effect . Inactivation was slowed down by D-lactate, a competitive inhibitor, and completely prevented by enzyme reduction . While no enzyme chemical modification could be demonstrated for the first step, the inactivation event of the second step could be ascribed to alkylation of a histidine belonging to proteolytic fragment beta of the enzyme . The only histidine present in the fragment sequence is His-373 . In the enzyme three-dimensional structure {Xia et al . (1987) Proc . Natl Acad . Sci . USA 84, 2629-2633} His-373 is well located, close to the cofactor, to play the role of the active-site base required by the chemical mechanism . Alternative chemical interpretations of the kinetic scheme are discussed, so is the difference between flavocytochrome b2 inactivation by fluoropyruvate and bromopyruvate.

Eur J Biochem, 1988 Apr 5, 173(1), 27 - 34
Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600 . Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A; Freist W et al.; For discrimination between isoleucine and 19 other amino acids by isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from kcat and Km values in amino-acylation of cognate tRNA(Ile)-C-C-A . Factors D are also products of initial discrimination factors I' and proof-reading factors II'; D = I' II' . Factors II' were calculated from AMP formation stoichiometries and factors I' as quotients of D and II'; I' = D/II' . II' is considered as a product of a pre- and post-transfer proof-reading factor (II' = II1II2), I' as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination . With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D = 38,000); other D values in a high range (10,000-20,000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300-700); the remaining amino acids are discriminated with medium D values (1000-10,000) . Discrimination factors D observed for isoleucyl-tRNA synthetase from E . coli are on average 2-3 times higher than for the yeast enzyme . Highest values were found in discrimination against Gly, Ala and Val (60,000-72,000), the lowest values for Cys, Arg and Trp (600-3000); the other amino acids exhibit D values between 20,000 and 50,000 . Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.

Biochem J, 1988 Apr 1, 251(1), 135 - 9
The unfolding and refolding of glutamate dehydrogenases from bovine liver, baker's yeast and Clostridium symbosium; West SM et al.; The unfolding behaviour of the hexameric glutamate dehydrogenases from bovine liver, Clostridium symbosium and baker's yeast in solutions of guanidinium chloride (GdnHCl) was studied . Changes in Mr studied by light-scattering indicate that, in each case, the hexamer dissociates to form trimers, which then dissociate to monomers at higher concentrations of GdnHCl . Dissociation to trimers is accompanied by a reversible loss of enzyme activity, but no gross structural changes can be detected by fluorescence or c.d . Dissociation to monomers is accompanied by large structural changes, and the loss of activity cannot be reversed by dilution . The parallel behaviour of all three enzymes shows that the previously noted inability of the isolated subunits of the bovine liver enzyme to refold {Bell & Bell (1984) Biochem . J . 217, 327-330} is not a result of any modification of the enzyme as a result of import into mitochondria, since the C . symbosium and baker's-yeast enzymes do not undergo any such post-translational translocation.

Eur J Biochem, 1988 Mar 15, 172(3), 633 - 9
Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast; Heidlas J et al.; Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, {hereafter called (S)- and (R)-enzymes} have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography . The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH . The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively . The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000 . Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively . The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme . Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively . Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.

J Biochem (Tokyo), 1988 Mar, 103(3), 463 - 9
X-ray study of baker's yeast lipoamide dehydrogenase at 4.5 A resolution by molecular replacement method; Takenaka A et al.; The molecular structure of lipoamide dehydrogenase from baker's yeast has been determined at 4.5 A resolution by molecular replacement techniques using the known structure of human erythrocyte glutathione reductase as a starting model . The enzyme crystallizes in the space group P2(1)2(1)2(1) with a = 98.6(2), b = 162.0(2), c = 69.4(2) A . There is one molecule per asymmetric unit . The enzyme is a dimeric protein of identical subunits related by a local two-fold symmetry . Comparison of the tertiary structures between glutathione reductase and the present enzyme shows that the folding is almost the same except for the N and C termini, although some slight shortening or shifting of alpha-helices was found in the electron density map . FAD molecules are found at similar positions to those of glutathione reductase . Since the amino acid residues around FAD and NAD binding sites and at the reaction centers of the two enzymes are strongly conserved, the lipoamide dehydrogenase may catalyze the opposite reaction through a similar mechanism to that proposed for glutathione reductase . The newly found C terminus is located near the edge of a deep cave at the interface between the two subunits . These additional 18 residues form a narrow entrance to the cave, in which the long chain of the dihydrolipoyl moiety of lipoate acetyltransferase will be bound.

Biochimie, 1988 Feb, 70(2), 205 - 13
Non-essential role of lysine residues for the catalytic activities of aspartyl-tRNA synthetase and comparison with other aminoacyl-tRNA synthetases; Theobald A et al.; Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp) . It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding . Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates . The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered . These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase . Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases . Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase . When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E . coli can be completely inactivated for their catalytic activities; for E . coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated . The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Biochem, 1988, 20(10), 1125 - 32
Purification and structural comparisons of the cytosolic and mitochondrial fumarases from baker's yeast; Boonyarat D et al.; 1 . The cytosolic and mitochondrial fumarases (EC 4.2.1.2) from baker's yeast (Saccharomyces cerevisiae) have been purified to homogeneity . 2 . Subunit molecular weights for the cytosolic and mitochondrial isoenzymes were 53,000 and 48,000 respectively . 3 . Peptide maps obtained after digestion of the two isoenzymes with trypsin were almost identical but showed significant small differences . The same was true of peptide maps obtained after digestion with the glutamic acid-specific proteinase from S . aureus.

Folia Microbiol (Praha), 1988, 33(5), 377 - 85
Sterol composition of a delta 5,7-sterol-rich strain of Saccharomyces cerevisiae during batch growth; Novotny C et al.; Sterol composition was examined during batch growth on complex media containing ethanol, molasses or glucose as the carbon source . The molasses-grown cells exhibited a balanced sterol composition throughout growth, maintaining the proportion of ergosterol to 24:28-dehydroergosterol equal to 1.4 . The negative effect of glucose on sterol synthesis manifested itself by decreasing the accumulation of 24:28-dehydroergosterol and total sterols but not of ergosterol . Using ethanol as the sole carbon source, a large amount of 24:28-dehydroergosterol accumulated, partly at the expense of other sterols . The gradual addition of nitrogen source during growth significantly decreased the accumulation of ergosterol, 24:28-dehydroergosterol and of total sterols . A general scheme of regulation of sterol synthesis in baker's yeast is presented.

Biomed Biochim Acta, 1988, 47(8), 731 - 6
Studies on the fast reacting thiol groups in phosphofructokinase from baker's yeast; Bar J et al.; The fast reacting thiol groups of yeast phosphofructokinase were studied by means of stopped-flow measurements . The enzyme was found to contain four very fast reacting cysteinyl residues determined by their reactivity towards 5,5'-dithiobis(2-nitrobenzoic acid) . A second class of eight thiol groups reveals an apparent first order rate constant which is three orders of magnitude lower than the rate constant of the first one . Due to the extreme high reactivity of the first class of cysteinyl residues partial oxidation was already observed under aerobic conditions . Fructose 6-phosphate, fructose 1.6-bisphosphate, and fructose 2.6-bisphosphate, respectively, decrease the reactivity of the first class of thiol groups but not the total number of the accessible cysteins . This result is discussed with regard to conformational changes of the enzyme after binding of the sugar phosphates.

Folia Haematol Int Mag Klin Morphol Blutforsch, 1988, 115(5), 669 - 87
{Effect of ultraviolet irradiation of autologous blood on cell volumes, cell adhesion and phagocytosis in normal probands and patients with multiple sclerosis}; Mix E et al.; UVB induced changes of blood cell properties were investigated in 12 MS patients and in 10 healthy volunteers serving as normal controls . The mean cell volume (MCV) was determined by electronic sizing, the granulocyte and lymphocyte adherence was estimated in a capillary assay, and the phagocytic activity of granulocytes was measured in a test system based on the incorporation of opsonized baker's yeast (Saccharomyces cerevisiae) . In MS patients the MCV of red cells and lymphocytes decreased rapidly within 6 UVB treatments . In contrast, the reduction of the granulocyte volume was delayed (between the 6th and 12th UVB) . In the control group the mean value of the red cell and lymphocyte MCV remained rather unaffected . There was a slight rise of the granulocyte volume after the 6th UVB . The only significant change of adherence was an increase of granulocyte adherence in MS patients . Untreated patients had a significantly enhanced phagocytic activity in comparison to the control group . 6 UVB treatments included a significant reduction of the phagocytic activity in MS patients . However, subsequently the percentage of phagocytizing cells increased again, whereas the particle uptake per cell continued to decrease . In the control group only minor UVB induced changes of phagocytosis were observed . The in vitro UV irradiation caused an enhanced phagocytosis in the majority of cases in both controls and MS patients . In general, under the UVB treatment all parameters examined changed in the sense of a normalisation, in that the measured values reached a new level lying between the extreme pretreatment values accompanied by a reduced standard deviation . The effect of UVB was more pronounced in MS patients when compared with normal controls . This could result from an enhanced sensitivity to the influence of UVB of pathologically altered cells in MS patients . The monitoring of the MCV of red cells and lymphocytes as well as the repeated testing of granulocyte phagocytosis are recommended for supportion of therapy planning and follow-up of MS patients.

Eur J Biochem, 1987 Dec 15, 169(3), 539 - 46
Amino-acid sequence of the cytochrome-b5-like heme-binding domain from Hansenula anomala flavocytochrome b2; Haumont PY et al.; Flavocytochrome b2 (L-lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains . Its first 100 residues constitute the heme-binding core, which is homologous to cytochrome b5 {B . Guiard, O . Groudinsky & F . Lederer (1974) Proc . Natl Acad . Sci . USA 71, 2539-2543} . We report here the amino acid sequence of the heme-binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2 . The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C . As isolated, the domain consists of 84 residues without any sulfur amino acids . It shows 49 identities with the heme-binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5 . Using the recently published three-dimensional structure of S . cerevisiae flavocytochrome b2 {Z-x . Xia, N . Shamala, P . H . Bethge, L . W . Lim, H . D . Bellamy, N . H . Xuong, F . Lederer and F . S . Mathews (1987) Proc . Natl Acad . Sci . USA 84, 2629-2633}, it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates . The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex {F . R . Salemme (1976) J . Mol . Biol . 102, 563-568}.

Arch Biochem Biophys, 1987 Dec, 259(2), 296 - 304
Epidermal growth factor receptor tyrosine kinase phosphorylation of glucose-6-phosphate dehydrogenase in vitro; Napier MA et al.; The specific tyrosine phosphorylation of glucose-6-phosphate dehydrogenase (G6PDH) by the epidermal growth factor (EGF) receptor in vitro is demonstrated . The Km values of the substrate G6PDH and of ATP for the receptor tyrosine kinase were ca . 1 and 10 microM, respectively . The rate of phosphorylation was EGF dependent, with a four-fold increase in Vmax in the presence of EGF . The phosphorylation was stimulated maximally by 0.2 microM or greater EGF, with an ED50 of ca . 20 nM which is consistent with the affinity of the solubilized receptor for EGF . Using conditions of 5 microM G6PDH, 100 microM ATP, 5 mM Mg2+, and 1 mM Mn2+, up to 0.3 mol phosphate was incorporated into 1 mol of the 55-kDa subunit of Baker's yeast G6PDH . Tryptic peptide mapping revealed several unique phosphopeptides for both Baker's yeast and bovine adrenal G6PDH . The patterns of phosphopeptides for a given enzyme were identical for basal and EGF-stimulated phosphorylation.

J Biochem (Tokyo), 1987 Dec, 102(6), 1531 - 7
Yeast calmodulin: structural and functional differences compared with vertebrate calmodulin; Luan Y et al.; Calmodulin of the baker's yeast (Saccharomyces cerevisiae) showed a similar affinity for Ca2+ to that of vertebrate calmodulin . The maximum binding number of Ca2+ to yeast calmodulin was, however, 3 mol/mol, which is lower than that of vertebrate calmodulin (4 mol/mol) . The same maximum activity of porcine brain phosphodiesterase was attained when 100 times higher concentration of yeast calmodulin than that of vertebrate calmodulin was added . On the other hand, the maximum activation of chicken gizzard myosin light chain kinase was attained with 1,000 times higher concentration of yeast calmodulin than that of vertebrate calmodulin, and the maximum activity with yeast calmodulin was less than 1/5 of that with vertebrate calmodulin . Several amino acid substitutions observed in the yeast calmodulin, particularly at the alpha-helical rod connecting the two globular domains, may affect the interaction mode of various target enzymes with this calmodulin.

Eur J Biochem, 1987 Nov 16, 169(1), 33 - 9
Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600 . Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A(3'NH2); Freist W et al.; For discrimination between isoleucine and the other 19 naturally occurring amino acids by isoleucyl-tRNA synthetases from baker's yeast and from Escherichia coli MRE 600 discrimination factors have been determined from kcat and Km values in aminoacylation of the modified tRNA(Ile)-C-C-A(3'NH2) . Discrimination factors D1 are products of an initial discrimination factor and a proof-reading factor: D1 = I1.II1 . From discrimination factors and AMP formation stoichiometry factors I1 and II1 were calculated . D1 values obtained with the enzyme from E . coli are generally higher than those observed with the yeast enzyme, in some cases up to ten times . With both enzymes low D1 values are found for cysteine, valine, and tryptophan (20-200), the highest values for glycine, alanine, and serine (600-4000) . I1 values calculated for the E . coli enzyme are slightly higher (4-145) than the factors observed with the yeast enzyme (1-85), proof-reading factors II1 of the E . coli enzyme are scattering about a mean value about 70, those of the yeast enzyme about a mean value about 50 . Initial discrimination factors I1 are directly related to hydrophobic interaction forces between the substrates and the enzymes . Plots of Gibbs free energy differences calculated from these factors are linearly related to the accessible surface areas of the amino acids . A hypothetical model of the binding site can be given in which selection of amino acids is achieved by hydrophobic forces and removal of steric hindrance.

Z Naturforsch {C}, 1987 Nov-Dec, 42(11-12), 1159 - 64
{Stereoisomeric aromatic compounds XIX: Asymmetric reduction of 4(5)-oxocarboxylic acids with baker's yeast}; Gessner M et al.; Asymmetric reduction of 4(5)-oxocarboxylic acids (esters) by baker's yeast and cyclization in acidic media yields optically active gamma(delta)-lactones . The evaluation of their chirality and optical purity was carried out by HPLC (HRGC) analysis of the corresponding 1,4(1,5)-diols via diastereomeric esters with (R)-Mosher acid (MTPA) and (S)-O-acyllactic acids respectively . By increasing the 4(5) alkyl side chain 4R(5R) configurated gamma(delta)-lactones with high ee-values are generated.

J Biochem (Tokyo), 1987 Oct, 102(4), 741 - 53
DNA polymerase A and its stimulative factor of baker's yeast: purification and characterization; Takizawa N et al.; DNA polymerase A (I or major) and its stimulative factor were purified from 15-20 kg wet weight of baker's yeast by several procedures, which were varied in order to examine the possible occurrence of proteolysis . The extraction was carried out in the presence of 10 or 3 mM phenylmethylsulfonyl fluoride (PMSF), followed by either batchwise adsorption-elution or column chromatography on DEAE-Sepharose (rapid or time-consuming, respectively) . These early steps were followed by column chromatographies on DEAE-, CM-, and heparin-Sepharoses, phosphocellulose, and Sephacryl S-300 . Preparations of the polymerase obtained by all the procedures described above showed a single protein band at Mr of about 145,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), unless they had been treated with 2-mercaptoethanol (ME) . After ME treatment, however, they showed two protein bands at Mr of about 145,000 and 75,000 in SDS-PAGE, except for those obtained by the procedure involving 10 mM PMSF and the batchwise adsorption-elution . All the preparations described above showed practically the same specific activity . This indicates that in intact cells, the polymerase consisted of a single peptide with Mr of about 145,000, and that after cell disruption, it was artificially hydrolyzed in a limited fashion into two peptides with Mr of about 75,000, which were still active and were linked to each other through a disulfide bond . Preparations of the factor obtained by all the procedures described above showed a single protein band at Mr of about 20,000 in SDS-PAGE before and after ME treatment . The relative activities of the purified polymerase were (100%), 123, 21, 37, 196, and 38% with native and denatured salmon sperm DNA, native and denatured calf thymus DNA, poly(dA-dT), and poly(dA).oligo(dT)10, respectively . With the addition of the purified factor, they were 173, 272, 173, 217, 173, and 247%, respectively, i.e., significantly stimulated . The purified factor also stimulated the activity of calf thymus DNA polymerase alpha by 150% with denatured salmon sperm DNA; Km was about 5 X 10(-10)M, practically the same as that of yeast DNA polymerase A . However, it hardly influenced the activities of Escherichia coli enzyme I or Micrococcus luteus enzyme.

J Biochem (Tokyo), 1987 Oct, 102(4), 705 - 14
Regulation of reductive production of succinate under anaerobic conditions in baker's yeast; Muratsubaki H; When baker's yeast grown aerobically on ethanol as a carbon source was anaerobically cultured in a medium containing glucose, the activity of a cytoplasmic fumarate reductase irreversibly catalyzing the conversion of fumarate to succinate increased, reaching about 3 times the original activity after 12 h, while the activity of succinate dehydrogenase was almost lost after 10 h . These results indicate that the citrate cycle is partially modified to become a reductive pathway leading to succinate during the anaerobic cultivation . In non-proliferating cells grown anaerobically on glucose, the rates of accumulating succinate and pyruvate were decreased and increased, respectively, with increasing concentrations of L-aspartate or NH4Cl in the medium containing glucose as a substrate . These changes were accompanied with increase in the cellular content of aspartate, an inhibitor of pyruvate carboxylase that is involved in supplying the intermediates of the citrate cycle, and pyruvate, a substrate of the enzyme . The aminotransferase inhibitor, aminooxyacetate, prevented the changes in succinate accumulation and cellular aspartate following the addition of NH4Cl . The addition of L-glutamate caused a marked increase in the rate of succinate accumulation without changing the cellular content of aspartate . Neither L-glutamate nor L-aspartate had the ability to produce succinate . The rate of glucose consumption was not changed upon adding these nitrogen compounds . Similar findings were also observed in experiments using proliferating cells . This report presents evidence that in cells containing a large amount of the fumarate reductase, the production of succinate from glucose is regulated by the cellular level of aspartate through the pyruvate carboxylase reaction and that glutamate regulates the succinate production by a mechanism distinct from that involved in the regulation by L-aspartate.

Biochim Biophys Acta, 1987 Aug 13, 925(2), 234 - 7
Activation of serine sulphydrase from baker's yeast (Saccharomyces cerevisiae) by vanadate; Meisch HU et al.; Vanadate stimulates the liberation of H2S from cysteine in intact cells of baker's yeast (Saccharomyces cerevisiae) with a maximal increase of 60% at 10 microM NH4VO3 . Protein separation from crude yeast extract yielded two active protein fractions which were found to catalyze the degradation of cysteine to H2S, pyruvate and ammonia or H2S and serine, respectively, thus characterizing them as cysteine desulphydrase and serine sulphydrase . Only the latter enzyme was found to be activated by vanadate, showing optimal enhancement of about 100% at 10 microM NH4VO3.

Biochem J, 1987 Jul 15, 245(2), 525 - 30
Denaturation and renaturation of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe; Johnson CM et al.; The denaturation by guanidinium chloride of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe was studied . The loss in activity broadly parallels the changes in protein structure detected by fluorescence and c.d . Renaturation can be brought about by dilution of the denaturing agent . These processes were compared with those in the enzymes from baker's yeast and rabbit muscle, which are tetrameric and dimeric respectively . The effects of the cofactor 2,3-bisphosphoglycerate on the structure and stability of the S . pombe enzyme were also investigated.

Biochem J, 1987 Jun 15, 244(3), 579 - 84
Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker's yeast); Kundu M et al.; The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity . Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity . This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside . Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity . Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection . Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin . Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity . However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site . In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.

N Engl J Med, 1987 May 21, 316(21), 1306 - 9
Enzyme-substitution therapy with the yeast Saccharomyces cerevisiae in congenital sucrase-isomaltase deficiency; Harms HK et al.; Sucrase-isomaltase deficiency is an inherited disaccharidase deficiency that leads to malabsorption of sucrose, with resulting diarrhea and abdominal distention and cramps . We investigated the sucrose-splitting effect of viable yeast cells in eight children with congenital sucrase-isomaltase deficiency, by means of the sucrose hydrogen breath test . This test is based on the fact that hydrogen is released from the malabsorbed sucrose by the colonic microflora . We found that 0.3 g of lyophilized Saccharomyces cerevisiae, given after loading with 2 g of sucrose per kilogram of body weight, reduced hydrogen excretion in all patients, on average by 70 percent, in parallel with a complete loss or evident reduction of clinical symptoms . In vitro, lyophilized and fresh S . cerevisiae (fresh baker's yeast) had appreciable sucrase activity, a low isomaltase and maltase activity, and virtually no lactase activity . The sucrase activity was more inhibited by undiluted than by diluted gastric juice . We conclude that patients with congenital sucrase-isomaltase deficiency who intentionally or unintentionally consume sucrose can ameliorate the malabsorption by subsequently ingesting a small amount of viable yeast cells, preferably on a full stomach.

Proc Natl Acad Sci U S A, 1987 May, 84(9), 2629 - 33
Three-dimensional structure of flavocytochrome b2 from baker's yeast at 3.0-A resolution; Xia ZX et al.; The structure of flavocytochrome b2 from baker's yeast was solved at 3.0-A resolution by the multiple isomorphous replacement method combined with solvent leveling procedures, using data collected from an area detector . The tetramer of Mr 230,000 has 4-fold symmetry . Each subunit contains a cytochrome domain consisting of the first 100 residues, a flavin-binding domain containing the next 386 residues, and an extended C-terminal tail of 25 residues . The cytochrome domain closely resembles microsomal cytochrome b5, whereas the flavin-binding domain contains a parallel beta 8/alpha 8 barrel motif similar to glycolate oxidase and trimethylamine dehydrogenase . Two of the four cytochrome domains are disordered in the crystals . The flavin ring and heme group are separated by about 16 A between their centers, and their planes are inclined by about 17 degrees to each other.

Biochim Biophys Acta, 1987 Apr 29, 908(3), 203 - 13
Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe; Broker M et al.; Recombinant plasmids were constructed that direct the synthesis of human antithrombin III in baker's yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe . The signal sequence of antithrombin III was recognized by both yeast species, and antithrombin III was secreted into the medium . When the signal sequence was replaced by a sequence of ten arbitrary amino acids, the product expressed from such a construct stayed inside the cell . Antithrombin III was glycosylated by the baker's and fission yeast and was immunologically identical to antithrombin III isolated from human plasma . Antithrombin III isolated from the culture media of recombinant yeasts was biologically active, as could be shown by progressive inhibitor activity and heparin cofactor activity.

Nucleic Acids Res, 1987 Mar 11, 15(5), 1887 - 904
Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae; Weygand-Durasevic I et al.; We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast . In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase . Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment . Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS . The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight . The codon usage of serS is typical of abundant yeast proteins . Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon . Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.

Braz J Med Biol Res, 1987, 20(5), 573 - 4
A test for the evaluation of ingestion by neutrophils; Pessoa MH et al.; A rapid, simple and inexpensive technique was developed to evaluate particle ingestion by neutrophils (PMNs) and alternatively investigate the activity of the complement system . Whole blood specimens, obtained in EDTA, were mixed with a suspension of activated Baker's yeast in minimal volumes (3 and 1 drops of each, respectively) . The mixture was incubated at 37 degrees C for 1 h and centrifuged at 200 g for 5 min . The sediment was resuspended in FBS and blood films were stained with Giemsa . Two hundred PMNs/film were counted and the percentage of phagocytizing cells and the mean number of ingested particles per cell were determined . Samples from 11 male and 15 female subjects showed mean rates of 80.0 +/- 10.8 (SD) and 83.3 +/- 5.8 of PMNs with phagocytic vacuoles containing 2.9 +/- 0.5 and 3.2 +/- 0.7 ingested particles/cell, respectively . There were no statistically significant differences between sexes.

Folia Microbiol (Praha), 1987, 32(3), 206 - 10
Effect of ammonium ions on delta 5,7-sterol synthesis in Saccharomyces cerevisiae; Novotny C et al.; The effect of ammonium concentration in the medium on delta 5,7-sterol synthesis was examined . Higher concentrations of this nitrogen source in the medium decreased sterol synthesis and accumulation during growth . An intermittent supply with ammonium resulted in a proportional synthesis of delta 5,7-sterols and biomass . The carbon to nitrogen molar ratio of greater than or equal to 40 allowed the maximum accumulation of delta 5,7-sterols with our strain of baker's yeast.

Genetika, 1987 Jan, 23(1), 21 - 9
{Detection of nucleotide sequences specific for retroviruses in Saccharomyces cells}; Ratovitskii EA et al.; Restriction endonuclease cleavage analysis and blotting hybridization of nuclear DNA and RNA to cloned avian sarcoma and murine leukemia virus genes (pol, scr and abl) demonstrated the presence and expression in baker's yeast cells of retrovirus-specific sequences . The relationship exists between the pol-specific yeast sequences and Ty cloned fragments . The results obtained are discussed in the light of evolutionary role of retroviral genes in cell division control and transposition.

Basic Appl Histochem, 1987, 31(3), 255 - 71
NAD pyrophosphorylase from yeast chromatin . Purification and properties; Magni G et al.; NAD pyrophosporylase has been purified to homogeneity from baker's yeast . The purification procedure is relatively simple and consists of high salt extraction of enzyme activity, precipitation with polyethylenimine followed by ion exchange and by ligand chromatography separations . The final enzyme preparation is homogeneous as judged by a single Coomassie blue-stainable band when run on non-denaturing and denaturating polyacrylamide gels . The native enzyme shows a molecular weight of about 200,000 calculated by gel filtration and sucrose gradient centrifugation . The protein possess quaternary structure and is composed by four apparently identical Mr 50,000 subunits . The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm . Isoelectric point is 6.2 . Amino acid composition shows the presence of 28 half-cystine residues, in agreement with the results obtained by titrating the enzyme in denaturing conditions with Ellman's reagent upon previous incubation with sodium borohydride . NAD pyrophosphorylase is a glycoprotein containing 2% sugar, 2 moles of alkali-labile phosphate per enzyme mol, and 1 mol of adenine moiety per enzyme mol . Therefore the possibility that the enzyme is ADP-ribosylated exists . Km for ATP, NMN and NaMN are 0.11 mM, 0.19 mM and 5 mM respectively . Kinetic analysis reveals a behaviour which is consistent with an ordered sequential Bi-Bi mechanism . pH optimum is in the range 7.2-8.4.

J Chromatogr, 1986 Dec 26, 371, 71 - 81
Enzymatic kinetic analyses that employ high-performance liquid chromatography . Competition between orotate- and hypoxanthine/guanine-phosphoribosyltransferases for a common substrate; Chung SH et al.; Enzymatic assay procedures that employ high-performance liquid chromatography (HPLC) have been proven to be sensitive and versatile methods for accomplishing kinetic analyses of enzyme-catalyzed reactions, with nucleotides as substrates or products . Both orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) have been purified from Baker's yeast and analyzed kinetically using a modification of published HPLC procedures . Because these two enzymes exist in the cytosol of yeast and might compete for the limiting (approximately equal to 15 microM) concentration of phosphoribosyl alpha-1-pyrophosphate (PRibPP), we elected to examine both equilibrium and steady-state effects of one enzymatic reaction on the other with HPLC . First, under the condition of equivalent mass concentrations of OPRTase and HGPRTase, the initial rate of orotidine monophosphate synthesis and the equilibrium state were greatly affected by the presence of HGPRTase activity . In contrast, the presence of the OPRTase activity had no effect on the HGPRTase-catalyzed reaction under these conditions . Second, to examine a competition by these enzymes for PRibPP in vivo, we have established that the total activities (units/ml) of OPRTase and HGPRTase in yeast cell extracts were 740 units/ml and 450 units/ml, respectively (a 1.7:1 ratio) . These relative activities were then employed in an in vitro reaction competition analysis . The results were similar to the those obtained from experiments where equivalent OPRTase and HGPRTase activities were employed and reveal profound initial velocity and equilibrium effects of one reaction on the other . Thus a real competition between these enzymes for PRibPP may occur in the yeast cell cytosol, as determined by this unique HPLC competition assay procedure.

Biokhimiia, 1986 Dec, 51(12), 2010 - 29
{Transketolase: structure and mechanism of action}; Kochetov GA; The structure and mechanism of action of transketolase are reviewed, with the primary emphasis laid on the baker's yeast enzyme . The oligomeric structure of transketolase, the interaction of the coenzyme with the apoenzyme and the role of phosphate groups in the substrate interaction with the protein have been studied . The role of essential groups of apotransketolase in the binding of the coenzyme, substrates as well as in the catalytic act are described . The peculiarities of formation of the enzyme active center are discussed.

Biokhimiia, 1986 Nov, 51(11), 1908 - 18
{Functional carboxylic group in the active center of transketolase}; Kuimov AN et al.; Baker's yeast transketolase is rapidly inactivated in the presence of carboxylic group modifiers, i.e., 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K . This inactivation is due to modification of the carboxylic group in the enzyme active center . The essential groups localized in the two active centers of transketolase differ in the rate of modification; accordingly, the inactivation kinetics appears as biphasic . A complete loss of the enzyme activity occurs as a result of modification of one carboxylic group per enzyme active center . The pKa value of modifiable groups is equal to about 6.5 . This modification decreases by two orders of magnitude the affinity of the substrate for the active center . The carboxylic groups are not directly involved in the interaction with the substrates; their modification does not significantly affect the coenzyme binding . It is supposed that these groups are responsible for the deprotonation of the second carbon in the thiamine pyrophosphate thiazolium ring.

FEBS Lett, 1986 Sep 29, 206(1), 121 - 4
Studies on inorganic pyrophosphatase using imidodiphosphate as a substrate; Smirnova IN et al.; Baker's yeast inorganic pyrophosphatase has been found to catalyze Mg2+-dependent hydrolysis of imidodiphosphate yielding phosphate and amidophosphate . The reaction proceeds linearly in the presteady state . The catalytic constant is maximal at pH 9.0 and equals 0.5 min-1 . Kinetic titrations of the enzyme with imidodiphosphate and Mg2+ have provided direct evidence for the involvement of three Mg2+ per active site in the transition state of the pyrophosphatase reaction.

Biokhimiia, 1986 Sep, 51(9), 1465 - 75
{Comparative study of glyceraldehyde-3-phosphate dehydrogenases isolated from rabbit skeletal muscles and baker's yeast using cationic fluorescent probes}; Klichko VI et al.; The tetrameric molecule of glyceraldehyde-3-phosphate dehydrogenase possesses the ability to bind fluorescent probes of cationic nature (auramine O and acridine orange) outside the active center . The rabbit skeletal muscle and yeast enzymes share some common features, e.g., the conformational non-equivalency of subunits; two subunits per tetramer can bind auramine O; conformational changes caused by the binding of adenyl mononucleotides and involving the microenvironment of auramine O binding sites; the ability to bind the cationic probe at pH values typical for the maximal activity of the enzyme in the reaction of glyceraldehyde 3-phosphate oxidation . The yeast and rabbit muscle enzymes are distinguished in terms of localization of the probe binding sites with respect to the active center and/or in the nature of conformational changes induced by NAD+ binding . It was demonstrated that nicotinamide mononucleotide may serve as a co-enzyme in glyceraldehyde 3-phosphate oxidation catalyzed by yeast dehydrogenase; this reaction in inhibited by AMP.

Eur J Biochem, 1986 Aug 15, 159(1), 117 - 24
Interaction of enzymes involved in triosephosphate metabolism . Comparison of yeast and rabbit muscle cytoplasmic systems; Tompa P et al.; The affinity of baker's yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase was tested by using a fluorescence-probe technique with fluorescein isothiocyanate attached covalently to the enzymes . The dissociation constants of the enzyme-enzyme complexes, as well as the rate constants of association and dissociation, were determined . Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20 degrees C) . The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species . Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non-hybrid complexes indicates that this ancient characteristic is probably conserved during evolution . A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metabolism.

J Exp Zool, 1986 Jul, 239(1), 131 - 2
Fructose-6-phosphate is not a substrate for glucose-6-phosphate dehydrogenase; Jeffery J et al.; D-Fructose-6-phosphate was shown not to be a substrate for glucose-6-phosphate dehydrogenases (EC . 1.1.1.49) from human erythrocytes, bovine adrenal, rat liver, three yeasts (brewer's yeast, baker's yeast, and Candida utilis), and Leuconostoc mesenteroides . These findings contrast with those of G.M . Kidder (J . Exp . Zool., 226:385-390, '83).

Eur J Biochem, 1986 Jul 1, 158(1), 149 - 57
Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases . Role of H+ gradient; de Meis L et al.; A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed . In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM . For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4 . This was not observed with the use of R . rubrum chromatophores . With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM . There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium . In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8 . For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively . In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively . With R . rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured . In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R . rubrum were practically the same . When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.

Biochemistry, 1986 Jun 17, 25(12), 3725 - 9
Nicotinamide mononucleotide adenylyltransferase . Molecular and enzymatic properties of the homogeneous enzyme from baker's yeast; Natalini P et al.; Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract . The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations . The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels . The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation . The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits . The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm . The isoelectric point is 6.2 . Amino acid composition analysis shows the presence of 28 half-cystine residues . The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride . NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme . Therefore, the possibility that the enzyme is ADP-ribosylated exists . The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively . Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism . The pH optimum is in the range 7.2-8.4.

Biochem J, 1986 Jun 1, 236(2), 617 - 20
The susceptibility towards proteolysis of intermediates during the renaturation of yeast phosphoglycerate mutase; Johnson CM et al.; The renaturation of the tetrameric enzyme phosphoglycerate mutase from baker's yeast after denaturation in guanidinium chloride was studied . Three proteinases (trypsin, chymotrypsin and thermolysin) cause extensive loss of activity of samples taken during the early stages of refolding . As judged by SDS/polyacrylamide-gel electrophoresis, the proteinases cause substantial degradation of the polypeptide chain with no evidence for large quantities of fragments of Mr greater than 6500 . These data suggest that the early intermediates in the refolding, especially the folded monomer, possess a number of sites that are susceptible to proteolysis.

Immunology, 1986 May, 58(1), 111 - 5
Evaluation of C3b/C3bi opsonization and chemiluminescence with selected yeasts and bacteria using sera of different opsonic potential; Turner MW et al.; C3 fragment opsonization and chemiluminescence were assessed using two yeasts (Saccharomyces cerevisiae and Candida albicans) and two strains of bacteria (Staphylococcus aureus and Escherichia coli) following incubation with sera previously shown to be either efficient or poor in the opsonization of baker's yeast . Sera with poor function consistently fixed lower levels of C3 fragments and generated less chemiluminescence with all four organisms than sera with normal function . The differences were most marked with the two yeasts . We conclude that sera with normal complement function but manifesting a relatively common defect in the opsonization of baker's yeast have a general opsonic defect.

Mutat Res, 1986 May, 165(3), 129 - 37
The isolation and characterization of an alkylating-agent-sensitive yeast mutant, ngs1; Nisson PE et al.; We have isolated and characterized a mutant of baker's yeast, Saccharomyces cerevisiae, carrying the new mutation, ngs1, which is sensitive to the toxic effects of monofunctional alkylating agents, but normal with respect to 254-nm ultraviolet light sensitivity . ngs1 mutants exhibited more or less normal reversion frequencies for his1-7 and ilv1-92 induced by each of these mutagens . The various sensitivities associated with ngs1 cosegregated and have been shown to be the result of a lesion in a single nuclear gene . Extracts of ngs1 and NGS1+ strains contained approximately equal levels of an activity that removes 3-methyladenine (3MA) and 7-methylguanine (7MG) from DNA in vitro . The mutation also depressed sporulation.

J Immunol, 1986 May 1, 136(9), 3189 - 97
Selective phagocytosis of gram-positive bacteria and interleukin 1-like factor production by a subpopulation of large granular lymphocytes; Abo T et al.; There has been a consensus that a large granular lymphocyte (LGL) population with natural killer (NK) function is nonadherent and nonphagocytic . However, a significant proportion of the nonadherent cells purified by the two-step depletion of adherent cells with a plastic surface and nylon wool columns engulfed Sta . aureus into their cytoplasm . These cells were morphologically identified as LGL in light and electron microscopies . Two-color immunofluorescence tests, furthermore, demonstrated that Leu-11+ LGL, Leu-11+7-, and Leu-11+7+, but not Leu-11-7+, phagocytosed Sta . aureus . Among the particles tested here, only Gram(+) bacteria were preferentially phagocytosed, whereas Gram(-) bacteria, other large-sized microbes (e.g., baker's yeast and Candida albicans), latex, silica, and carbonyl iron were not . LGL exhibited a substantial level of bactericidal activity against Sta . aureus, although the level was one third of that mediated by monocytes . When Gram(+) bacteria were incubated with nonadherent cells for 18 hr, significant amounts of interleukin 1 (IL 1)-like factors (or IL 1 itself) as well as interferon were detected in the supernatants . On the other hand, this incubation did not induce interleukin 2 (IL 2) . The IL 1-like factor producer cells were demonstrated to be the low-density lymphocytes on Percoll separation and to have the Leu-11+ phenotype . The phagocytosis was suggested to be an important stimulus in producing IL 1-like factors from LGL . Thus, the treatment of cells with cytochalasin B, a microfilament disrupting agent, completely abrogated both phagocytosis and IL 1-like factor production . Some cell wall components of Gram(+) bacteria might be important to a recognition process of the phagocytosis, since the protoplasts of Sta . aureus, when prepared by the treatment of bacteria with lysostaphin, were no longer phagocytosed by LGL . The present results therefore identify an additional unique characteristic similar to, but not identical with, the myelomonocytic nature of Leu-11+ LGL.

J Protozool, 1986 May, 33(2), 186 - 91
Cross-reactive polysaccharides from Trypanosoma cruzi and fungi (especially Dactylium dendroides); Schnaidman BB et al.; Cross-reactivity between fungal and Trypanosoma cruzi polysaccharides, owing to common residues of beta-D-galactofuranose, beta-D-galactopyranose, and alpha-D-mannopyranose, was demonstrated by using rabbit immune sera against T . cruzi epimastigotes and sera from patients with Chagas' disease . Several chagasic (Ch) sera precipitated partly purified galactomannans from Aspergillus fumigatus and from T . cruzi epimastigotes and also the galactoglucomannan from Dactylium dendroides . Reaction of one Ch serum with T . cruzi galactomannan (GM) was completely inhibited by synthetic beta-D-Galf-(1----3)-Me alpha-D-Manp, and that of another Ch serum with a purified D . dendroides galactoglucomannan (GGM) was partly inhibited by (1----6)-linked (81%) or by (1----3)-linked (33%) beta-D-Galf-Me alpha-D-Manp . The beta-D-Galf-(1----3)-alpha-D-Manp epitope was present in both T . cruzi and D . dendroides polysaccharides . Rabbit anti-T . cruzi antisera precipitated A . fumigatus GM, T . cruzi antigenic extracts containing the lipopeptidophosphoglycan (LPPG), T . cruzi alkali-extracted GM, a synthetic GM, and D . dendroides GGM . Weak reactivities were obtained for a Torulopsis lactis-condensi GM containing beta-D-Galp terminal residues and for baker's yeast mannan with alpha-D-Manp-(1----3)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----2) side chains . An anti-LPPG rabbit serum precipitated D . dendroides GGM--a reaction inhibited (82%) by beta-D-Galf-(1----3)-Me alpha-D-Manp and . less efficiently, by a (1----5)-linked beta-D-Galf-tetrasaccharide . Sera from mice immunized with D . dendroides whole cells reacted with CL-strain trypomastigotes as shown by indirect immunofluorescence, by a Staphylococcus adherence test, but were not lytic . Mice immunized with D . dendroides were not protected against a challenge with virulent T . cruzi trypomastigotes.

J Chromatogr, 1986 Apr 11, 376, 141 - 8
Affinity partitioning: a new approach for studying dye-protein interactions; Kopperschlager G et al.; Affinity partitioning in an aqueous two-phase system composed of dextran and dye-liganded polyethylene glycol was applied to the investigation of the affinity of phosphofructokinase and glucose-6-phosphate dehydrogenase from baker's yeast, as well as of albumin and prealbumin from human serum to diverse reactive dyes . From the change in the partition coefficient K of the proteins in the two-phase system in the presence and in the absence of the dye-liganded polymer, expressed as delta log K, quantitative data for the maximal extraction power and for the affinity of the proteins to various reactive dyes were obtained . The affinity partitioning effect on prealbumin is markedly increased by an excess of monomeric albumin . This points to an interaction of the two proteins in the presence of the dye, Remazol Yellow GGL . The competitive effect of various natural ligands on binding reactive dyes to proteins can be investigated by means of affinity phase partitioning as demonstrated on phosphofructokinase and prealbumin.

Biol Chem Hoppe Seyler, 1986 Apr, 367(4), 331 - 41
Isoleucyl-tRNA synthetase from baker's yeast . Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine; Freist W et al.; The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast . For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively . At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered . With the noncognate substrates valine and tRNAVal such differences could not be detected . The substrate ATP did not show any change of its Km value as far as the reaction was measurable . Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one . In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine . From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined . In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant . The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism . Two hypothetical catalytic cycles are postulated.

Eur J Biochem, 1986 Feb 17, 155(1), 111 - 9
The complete amino acid sequence of adenylate kinase from baker's yeast; Tomasselli AG et al.; The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP----MgADP + ADP) from baker's yeast has been determined . Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively . They were sequenced with either a solid-phase sequencer or a gas-phase sequencer . Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by endoproteinase Lys-C and cyanogen bromide cleavages . The N-terminus is blocked by an acetyl group as shown by proton magnetic resonance . Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2-iodosobenzoic acid cleavages . The enzyme is a monomer of 220 amino acids with Mr 24077 . Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putative active-site region of the enzyme . After position 111, however, there is an insertion of 32 residues in the yeast species, similar to the adenylate kinase and the GTP:AMP phosphotransferase from beef heart mitochondria.

J Biochem (Tokyo), 1986 Feb, 99(2), 601 - 4
Improved purification of Aspergillus oryzae 1,2-alpha-mannosidase by using mannan gel affinity chromatography; Tanimoto K et al.; In order to facilitate the purification of 1,2-alpha-mannosidase from an enzyme product of Aspergillus oryzae, we have devised a rapid and simple procedure . A partially purified enzyme preparation obtained from the A . oryzae enzyme product, by means of ammonium sulfate fractionation followed by CM-Sephadex C-50 chromatography, was subjected to affinity chromatography with baker's yeast mannan gel as an adsorbent . 1,2-alpha-Mannosidase was retarded and well separated from the major protein peak on the affinity column . After a second affinity chromatography under the same conditions, 1,2-alpha-mannosidase was finally purified 7,500-fold with a 22.9% yield . The enzyme preparation thus obtained was quite suitable for the structural analysis of glycoconjugates.

Ann Clin Res, 1986, 18(1), 65 - 8
Selenium yeast; Korhola M et al.; Baker's yeast is able to assimilate carbon, nitrogen, phosphorus and sulphur sources together with a great number of minerals and trace elements into a palatable, nutritious product . The metabolism of yeast is precisely controlled during the production growth phase and thus it is possible to determine the composition of the product by controlling the raw materials . Because of existing deficiencies in the availability of certain trace elements, mainly selenium, in Finnish diets, we started testing the possibilities for enriching yeast with this essential trace element about five years ago . We have succeeded in developing a special yeast product with a selenium concentration of 500 mg/kg dry matter . Selenium was expected, because of its structural similarity to sulphur, to replace sulphur in the biosynthetic reactions of the yeast cell . We have recently studied the incorporation and distribution of selenium in yeast with radioactive selenium (75Se) . Analysis of the protein fraction of selenium yeast has shown that selenium is present in all the major soluble proteins . Selenomethionine was identified as the major selenium-containing compound in the protein fraction as well as in the whole cell.

Biomed Biochim Acta, 1986, 45(3), 273 - 80
Interactions of immobilized and free triazine dyes with glucose-6-phosphate dehydrogenase from yeast; Reuter R et al.; Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) prepared from baker's yeast binds to immobilized Cibacron Blue F3G-A and Procion Red HE-3B . In this paper the two dyes are compared with respect to their use in the purification of this enzyme . Cibacron Blue chromatography was found useful at an early stage of purification for the removal of contaminating hexokinase, phosphoglucose isomerase and phosphoglucomutase . With Procion Red HE-3B Sepharose the NADP dependent enzymes phosphogluconate dehydrogenase and glutathione reductase are separable from glucose-6-phosphate dehydrogenase . Unlike Cibacron Blue gel chromatography, the enzyme can be specifically eluted from Procion Red HE-3B Sepharose by a NADP gradient . Other monochlorotriazine dyes like Xirone Brillant Red BHD, 4BHD, 6BHD and GHD and the dichlorotriazine dye Procion Brown MX-5BR immobilized to Sepharose have only little binding affinity to glucose-6-phosphate dehydrogenase . The binding behaviour of different immobilized triazine dyes for pre-purified and purified glucose-6-phosphate dehydrogenase is compared . In addition, the influence of the free dyes on the activity of glucose-6-phosphate dehydrogenase is studied . It is demonstrated that the results of kinetic and binding studies with the purified enzyme are not uncritically applicable for the selection of a dye as ligand for affinity chromatography during enzyme preparation.

Nucleic Acids Res, 1985 Dec 9, 13(23), 8587 - 601
Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region; Struhl K; Genes of the baker's yeast Saccharomyces cerevisiae are densely clustered on 16 linear chromosomes . Here, I characterize a 1.8 kb region of chromosome XV containing the entire structural gene for the histidine biosynthetic enzyme imidazoleglycerolphosphate (IGP) dehydratase (his3) as well as the promoter sequences and 5'-proximal mRNA coding regions for the adjacent genes . The his3 gene encodes several mRNA species averaging 820 bases in length, all of which contain an open reading frame of 219 codons . The location of this open reading frame coincides with the his3 gene as defined by functional criteria, suggesting that the primary translation product of yeast IGP dehydratase has a molecular weight of 23,850 . Phenotypic analysis of mutations constructed in vitro indicate that one of the adjacent genes (pet56) is required for mitochondrial function, whereas the other gene (ded1) is essential for cell viability . The pet56 and his3 genes are transcribed divergently from initiation sites that are separated by only 192 bp . Transcription of the ded1 gene is initiated only 130 bp beyond the 3'-end of the his3 mRNA coding region . These results suggest that these unrelated genes are located extremely close together and that the spacer regions between them consist largely of promoter and terminator sequences.

Biochem Int, 1985 Dec, 11(6), 913 - 20
An investigation of the carboxyl group function in the active center of transketolase; Kuimov AN et al.; Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide . pKa of the modified carboxyl groups is approximately 6.5 . An investigation of the initial steps of enzymatic catalysis monitored by a changes in the circular dichroism spectra and in an oxidation reaction with ferricyanide made it possible to conclude that the modification interferes with the donor substrate attachment to the enzyme . Evidence obtained was suggesting that the carboxyl group of the active center facilitates dissociation of a proton from the carbon atom in the second position of the thiamine pyrophosphate thiazolium ring.

Eur J Biochem, 1985 Oct 15, 152(2), 419 - 28
Complete amino acid sequence of flavocytochrome b2 from baker's yeast; Lederer F et al.; Each subunit of baker's yeast flavocytochrome b2 can be selectively cleaved by proteases into two fragments, amino-terminal fragment alpha and carboxy-terminal fragment beta . The primary structure of the former has been reported before {Ghrir, B., Becam, A . M . & Lederer, F . (1984) Eur . J . Biochem . 139, 59-74} . The amino acid sequence of the 197-residue fragment beta has now been established . The fragment was cleaved with cyanogen bromide; the three peptides thus obtained were submitted to digestions with Staphylococcus aureus V8 protease, chymotrypsin and trypsin, sometimes after succinylation . The complete fragment was also submitted to tryptic cleavage after citraconylation . Peptides were separated by thin-layer finger-printing or high-pressure liquid chromatography . They were mostly sequenced in a liquid-phase sequenator . The 511-residue amino acid sequence of the mature protein is thus completely established . Secondary structure predictions indicate an alternation of helical and extended structure, with a higher percentage of the former . Comparisons with other flavoproteins do not detect any significant sequence similarity.

Arch Biochem Biophys, 1985 Sep, 241(2), 533 - 9
Purification and properties of an endo-alpha-mannan hydrolase from the mushroom Volvariella volvacea; Khowala S et al.; The enzyme, endo-alpha-mannanase, from culture filtrate of a mushroom Volvariella volvacea has been purified 73-fold by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex), and gel-permeation chromatographies on Bio-Gel P-300 and on Sephacryl S-200 columns . The enzyme preparation gave a single protein band on sodium dodecyl sulfate-disc gel electrophoresis at pH 6.8 and has a molecular weight of approx . 56,000 . It has no alpha- or beta-mannosidase activity and does not act on beta-gluco-or galactomannan . The enzyme shows maximum activity on baker's yeast alpha-mannan at pH 5.0 and at 55 degrees C, and is fairly stable between pH 3 and 6 and temperatures up to 50 degrees C . The Km is 32.25 mg mannan/ml . Enzyme activity is inhibited by Hg2+, sodium azide, iodoacetic acid, EDTA, and Ag+, in decreasing order.

Anal Biochem, 1985 Aug 15, 149(1), 248 - 60
Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase; Labbe P et al.; We describe fluorometric assays for two enzymes of the heme pathway, coproporphyrinogen oxidase and protoporphyrinogen oxidase . Both assays are based on measurement of protoporphyrin IX fluorescence generated from coproporphyrinogen III by the two consecutive reactions catalyzed by coproporphyrinogen oxidase and protoporphyrinogen oxidase . Both enzymatic activities are measured by recording protoporphyrin IX fluorescence increase in air-saturated buffer in the presence of EDTA (to inhibit ferrochelatase that can further metabolize protoporphyrin IX) and in the presence of dithiothreitol (that prevents nonenzymatic oxidation of porphyrinogens to porphyrins) . Coproporphyrinogen oxidase (limiting) activity is measured in the presence of a large excess of protoporphyrinogen oxidase provided by yeast mitochondrial membranes isolated from commercial baker's yeast . These membranes are easy to prepare and are stable for at least 1 year when kept at -80 degrees C . Moreover they ensure maximum fluorescence of the generated protoporphyrin (solubilization effect), avoiding use of a detergent in the incubation medium . The fluorometric protoporphyrinogen oxidase two-step assay is closely related to that already described (J.-M . Camadro, D . Urban-Grimal, and P . Labbe, 1982, Biochem . Biophys . Res . Commun . 106, 724-730) . Protoporphyrinogen is enzymatically generated from coproporphyrinogen by partially purified yeast coproporphyrinogen oxidase . The protoporphyrinogen oxidase reaction is then initiated by addition of the membrane fraction to be tested . However, when very low amounts of membrane are used, low amounts of Tween 80 (less than 1 mg/ml) have to be added to the incubation mixture to solubilize protoporphyrin IX in order to ensure optimal fluorescence intensity . This detergent has no effect on the rate of the enzymatic reaction when used at concentrations less than 2 mg/ml . Activities ranging from 0.1 to 4-5 nmol protoporphyrin formed per hour per assay are easily and reproducibly measured in less than 30 min.

Anal Biochem, 1985 Aug 1, 148(2), 542 - 5
Use of carboxypeptidase Y in studies of the mode of insertion of cytochrome b5 into lipid vesicles; Carlsen J et al.; Carboxypeptidase Y preparations from baker's yeast have been found to exhibit endopeptidase activity when cytochrome b5 was used as substrate . As the susceptibility of cytochrome b5 to attack by carboxypeptidase Y has been used to distinguish between two modes of insertion of cytochrome b5 into lipid bilayer, one which has the C terminal buried in the lipid bilayer and one which has a free C terminal, caution should be taken when employing carboxypeptidase Y preparations for this type of studies.

Mutat Res, 1985 Jun-Jul, 150(1-2), 211 - 6
New mutations affecting induced mutagenesis in yeast; Lawrence CW et al.; Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions . To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants . Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6 . The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process . The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction . The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

Arch Biochem Biophys, 1985 May 15, 239(1), 87 - 93
RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris; Weinstein JD et al.; Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase) . Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components . RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor . The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated . After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C . vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography . Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E . coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.

Z Naturforsch {C}, 1985 May-Jun, 40(5-6), 364 - 72
A small-angle X-ray scattering study on pre-irradiated malate synthase . The influence of formate, superoxide dismutase, and catalase on the X-ray induced aggregation of the enzyme; Zipper P et al.; The sulfhydryl enzyme malate synthase from baker's yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: congruent to 10 mg/ml; volume: 120 microliters), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase . After X-irradiation, a small aliquot of the irradiated solutions was tested for enzymic activity while the main portion was investigated by means of small-angle X-ray scattering . Additionally, an unirradiated sample without additives was investigated as a reference . Experiments yielded the following results: X-irradiation in the absence of the mentioned scavengers caused considerable aggregation, fragmentation, and inactivation of the enzyme . The dose Dt37 for total (= repairable + non-repairable) inactivation resulted as 4.4 kGy . The mean radius of gyration was found to be about 13 nm . The mean degree of aggregation was obtained as 5.7, without correction for fragmentation . An estimation based on the thickness factor revealed that about 19% of material might be strongly fragmented . When this amount of fragments was accordingly taken into account, a value of 7.1 was obtained as an upper limit for the mean degree of aggregation . The observed retention of the thickness factor and the finding of two different cross-section factors are in full accord with the two-dimensional aggregation model established previously (Zipper and Durchschlag, Radiat . Environ . Biophys . 18, 99-121 (1980)) . The presence of catalytic amounts of superoxide dismutase and/or catalase, in the absence of formate, during X-irradiation reduced both aggregation and inactivation significantly . The presence of formate (10 or 100 mM) during X-irradiation led to a strong decrease of aggregation and inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1985 Apr 8, 183(1), 21 - 4
Conditions of activation of yeast plasma membrane ATPase; Sychrova H et al.; The in vivo activation of the H+-ATPase of baker's yeast plasma membrane found by Serrano in 1983 was demonstrated with D-glucose aerobically and anaerobically (as well as in a respiration-deficient mutant) and, after suitable induction, with maltose, trehalose, and galactose . The activated but not the control ATPase was sensitive to oligomycin . No activation was possible in a cell-free extract with added glucose . The ATPase was not activated in yeast protoplasts which may account for the absence of glucose-stimulated secondary active transports in these wall-less cells and provide support for a microscopic coupling between ATPase activity and these transports in yeast cells.

Pharmazie, 1985 Apr, 40(4), 250 - 3
{The chemical composition of "zymosans" from yeasts}; Zimmermann G et al.; The chemical composition of "Zymosanes" (complex cell-wall-polysaccharides), prepared by different methods from Baker's yeast, were analyzed by classical and modern techniques, e.g . GLC and TLC . They are very complex natural products consisting of a basic structure of polysaccharides, which proteins and different lipids are bound to . Higher purified zymosanes seem to be free from nucleic acids and do not contain sialic acid.

J Biochem (Tokyo), 1985 Apr, 97(4), 1201 - 9
Characterization of fumarate reductase from baker's yeast: essential sulfhydryl group for binding of FAD; Muratsubaki H et al.; Fumarate reductase apoenzyme having the ability to reconstitute active enzyme was obtained by dialyzing the holoenzyme against 1 M KBr . The dissociation constant of the FAD-apoenzyme complex was 2.3 X 10(-8) M . The denatured holoenzyme and apoenzyme possessed seven sulfhydryl (SH) groups as determined with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) . In the native apoenzyme, five SH-groups reacted with DTNB, and four of them were completely protected by the addition of FAD, while in the native holoenzyme, one was modified without inactivation . These results indicate that one SH-group is located on the surface of the enzyme molecule, four at or near the FAD-binding site, and two deeply embedded in the molecule . The modification of the apoenzyme caused inhibition of binding of FAD, resulting in loss of the ability to reconstitute enzymatic activity . Analyses of the data by statistical and kinetic methods suggested that a reactive SH-group is involved among the four SH-groups in the binding of FAD to the apoenzyme.

Eur J Biochem, 1985 Apr 1, 148(1), 145 - 54
On the mechanism of flavin modification during inactivation of flavocytochrome b2 from baker's yeast by acetylenic substrates; Pompon D et al.; The reaction of 2-hydroxy-3-butynoate, a suicide substrate, with flavocytochrome b2 {F . Lederer (1974) Eur . J . Biochem . 88, 393-399} has been reinvestigated . It is shown that no inactivation occurs under anaerobic conditions . In the presence of ferricyanide, the partition ratio between oxidation and inactivation is 3200 . Ketobutynoate has no effect on oxidized flavocytochrome b2 . But it inactivates the reduced enzyme, while undergoing catalytic reduction to hydroxybutynoate . The partition ratio between reduction and inactivation is 5 . Inactivation followed by borohydride reduction was carried out in parallel with lactate oxidase and flavocytochrome b2 . The decomposition products of the initial adduct formed between flavin and inactivator were isolated and characterized . One of them (compound II) was obtained from both enzymes and is identical to the one previously isolated from hydroxybutynoate-inactivated lactate oxidase {Schonbrunn et al . (1976) Biochemistry 15, 1798-1807} . Its decarboxylated derivative (compound I) was also formed . Another major adduct, compound III, was isolated only from flavocytochrome b2 . Its structure and the conditions in which it appears suggest it is formed from the same primary adduct as compounds I and II, but by a different decomposition mode, on the enzyme itself . Altogether these results strengthen the idea that inactivation is caused by reaction between oxidized flavin and an allenic carbanion, the isomerisation product of a normal reaction intermediate . It is proposed that differences in the rate-determining step of the redox reaction explain the differences in the inactivation process which are observed between flavocytochrome b2, lactate oxidase and hydroxyacid oxidase.

Antibiot Med Biotekhnol, 1985 Feb, 30(2), 86 - 90
{Joint cultivation of Streptococcus lactis, LMU strain, and various species of yeasts}; Egorov NS et al.; When S . lactis, strain LMU and yeast were plated out simultaneously on media containing different amounts of amine nitrogen and vitamins, the yeast did not stimulate the synthesis of nisin . Plating out of S . lactis on media with grown cultures of the Rostov baker's yeast, American baker's yeast, Candida guilliermondii, Candida clausenii, Fabospora fragilis, Fabospora macedoniensis, Octosporomyces octosporus, Saccharomyces cerevisiae, races "Ya" and "M" and Saccharomyces globosus resulted in inhibition of the S . lactis growth and nisin synthesis . Cultivation of S . lactis on media with grown cultures of Saccharomyces ludwigii, Hansenula anomala, Kluyveromyces lactis, Endomyces magnusi, Zigowilliopsis californicus, and Rhodotorula colostri resulted in formation of an association providing active growth and development of S . lactis with complete realization of its biosynthetic capacity.

Histochemistry, 1985, 83(3), 251 - 5
Cytochemical localization of alkaline phosphatase in the cell membrane of Dictyostelium discoideum amebae; Glomp I et al.; We demonstrated that alkaline phosphatase was localized on the cell membrane of Dictyostelium discoideum amebae and on isolated plasma membranes . The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine . The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).

Radiat Environ Biophys, 1985, 24(2), 99 - 111
Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase . Effects of formate, superoxide dismutase, catalase, and dithiothreitol; Durchschlag H et al.; Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml) . The radiation induced changes of enzymic activity were registered at about 0, 30, 60 h after irradiation . To elucidate the role of OH., O-.2, and H2O2 in the X-ray inactivation of the enzyme, experiments were performed in the absence or presence of different concentrations of specific additives (formate, superoxide dismutase, catalase) . These additives were added to malate synthase solutions before or after X-irradiation . Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol . Experiments yielded the following results: Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Ar = 3% (corresponding to a D37 = 0.6 kGy), and led to further slow inactivation in the post-irradiation phase . Repairs, initiated at different times after irradiation, restored enzymic activity considerably . The repair initiated at t = 0 led to Ar = 21%; repairs started later on resulted in somewhat lower activities . The decay of repairability, however, was found to progress more slowly than post-irradiation inactivation itself . After completion of repair the activities of repaired samples did not decrease significantly . The presence of specific additives during irradiation caused significant protective effects against primary inactivation . The protection by formate was very pronounced (e.g., Ar = 72% and D37 = 6 kGy for 100 mM formate) . The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate . Both the presence of specific additives during irradiation and the addition of additives after irradiation may alter the post-irradiation inactivation . Catalase turned out to be the most potent inhibitor of post-irradiation inactivation; superoxide dismutase showed an ambivalent behaviour, it accelerated or impeded post-irradiation inactivation; formate, when added after irradiation, exhibited a moderate protective effect . The presence of specific additives, added before and/or after irradiation, influenced the repair behaviour to some extent . The highest activity achieved by repair amounted to about 90% of the activity of the corresponding unirradiated sample . The percentual gain of activity was found to be the greater the lower the residual activity of the enzyme was before initiation of repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Crit Rev Microbiol, 1985, 12(2), 131 - 51
alpha-Aminoadipate pathway for the biosynthesis of lysine in lower eukaryotes; Bhattacharjee JK; Bacteria and green plants use the diaminopimelate pathway for the biosynthesis of the essential amino acid, lysine; however, yeast and other higher fungi use the alpha-aminoadipate (AA) pathway . The AA pathway has been investigated in detail biochemically, genetically, and in terms of regulatory mechanisms in the baker's yeast Saccharomyces cerevisiae . The genetic analysis of lysine auxotrophs of S . cerevisiae revealed that there are more than 12 lysine genes for 8 enzyme-catalyzed steps . Lysine genes are not linked to each other and seven of the genes are mapped on six different linkage groups (chromosomes) . The gene-enzyme relationships have been determined for ten of the lysine loci which include two unlinked gene functions required for each of AA reductase (LYS2 and LYS5) and Saccharopine reductase (LYS9 and LYS14) . Five of the lysine enzymes are localized in mitochondria and three in cytosol . The lysine pathway of S . cerevisiae is regulated by feedback inhibition and end product repression . Two, and possibly three, of the enzymes exhibit general control of amino acid biosynthesis and at least five of the enzymes coded for, by unlinked genes, are simultaneously depressed in a regulatory (repressor) gene-mutant.

Ciba Found Symp, 1985, 111, 31 - 9
Enzyme-controlled reactions giving alkanols of use in the synthesis of biologically active molecules; Roberts SM; (+/-)-Bicyclo{3.2.0}hept-2-en-6-one was reduced using a variety of fungi including yeasts . Baker's yeast gave 6-exo-(1R,5S,6S)-bicyclo{3.2.0}hept-2-en-6-ol and 6-endo-(1S,5R,6S)-bicyclo{3.2.0}hept-2-en-6-ol while Curvularia lunata and Mortierella ramanniana gave only the 6-endo-alcohol and optically active bicycloheptenone . The optically active bicycloheptenols were used to prepare prostaglandin F2 alpha in the natural configuration.

Ciba Found Symp, 1985, 111, 112 - 27
Stereochemistry and synthetic applications of products of fermentation of alpha,beta-unsaturated aromatic aldehydes by baker's yeast; Fuganti C et al.; Baker's yeast fermenting on D-glucose converts 2-substituted C6-C3 alpha,beta-unsaturated aromatic aldehydes into the corresponding 3-phenylprop-2-en-1-ols and 3-phenylpropan-1-ols, and into the 4-substituted (2S,3R)-5-phenylpent-4-en-2,3-ols . The formation of the C6-C3 alcohols from the aldehydes by baker's yeast was already known, but the production of the methyl diols is new . The conversion of C6-C3 alpha,beta-unsaturated aldehydes into the C6-C5 methyl diols can be viewed as the overall consequence of two distinct chemical operations: (1) addition of a C2 unit equivalent to acetaldehyde onto the Si-face of the carbonyl carbon of the unsaturated aldehyde forms the (R)-alpha-hydroxy ketone in an acyloin-type condensation, and (2) reduction of this intermediate on the Re-face of the carbonyl gives the diol actually isolated . There is some tolerance by the enzymic system(s) involved in the reaction(s) leading from the C6-C3 alpha,beta-unsaturated aromatic aldehydes to the 4-substituted (2S,3R)-5-phenylpent-4-en-2,3-ols as far as the structure of the aromatic aldehydes and the substitutents in the alpha position are concerned, but acetaldehyde is the only aldehyde accepted as second terminus of the reaction . However, synthetic alpha-hydroxy ketones, prepared from aldehydes that cannot be directly converted by yeast into the corresponding methyl diols, are reduced by yeast . This indicates that the reason direct conversion of the aldehydes does not occur is that these materials probably cannot be accepted as substrates by the condensing enzyme(s) . The (2S,3R)-diols can be used instead of natural carbohydrates as starting materials for the synthesis of optically active forms of natural products belonging to different structural classes . Applications of these diols in the synthesis of L-daunosamine, the natural form of vitamin E and other products are discussed.

Dev Biol Stand, 1985, 59, 31 - 9
Reduction of proteolytic breakdown in microbial homogenates; Hedman P et al.; Proteolytic enzymes are present in many biological raw materials used for the production of proteins and peptides . Such enzymes are active not only in homogenates of bacteria and yeast but also in ascites liquid and cell culture media used for the production of monoclonals . A new protease substrate was used to determine protease activity in homogenates of E . coli and baker's yeast, which are useful for the production of therapeutically valuable polypeptides . Using this substrate we found that cooling and the addition of protease inhibitors had little effect on the proteolytic activity . Most of the proteolytic activity however could be adsorbed to hydrophobic gel media . Data is given on the protease adsorption at different temperatures and ionic strengths.

Folia Microbiol (Praha), 1985, 30(1), 90 - 1
Transport of ethanol in baker's yeast; Kotyk A et al.; Ethanol is transported into various strains of baker's yeast by simple diffusion (no effect of inhibitors and a linear concentration dependence of the initial rate of uptake and final distribution in cells) . It distributes itself in 96.6 +/- 16.2% of intracellular water.

Biomed Biochim Acta, 1985, 44(7-8), 1025 - 34
The use of progress curves for the estimation of inactivation rate constants of enzymes; Kuttner GA et al.; Progress curve analysis is applied to the estimation of inactivation rate constants . The heat inactivation of inorganic pyrophosphatase from baker's yeast was studied assuming a simple one-substrate mechanism containing two irreversible inactivation steps for the free enzyme and the enzyme-substrate complex, respectively . Four kinetic parameters including Km simultaneously may be detected from one progress curve . The temperature dependences derived from the estimated rate constants for several inactivating temperatures agree well with that obtained from preincubation measurements.

J Cyclic Nucleotide Protein Phosphor Res, 1985, 10(1), 121 - 7
Cyclic AMP phosphodiesterase activities in growing cells of baker's yeast (Saccharomyces cerevisiae); Suoranta K; The activities of two cyclic AMP phosphodiesterases of baker's yeast (Saccharomyces cerevisiae) were measured during diauxic batch growth on 2% glucose . The specific activity (units/mg of yeast protein) of the Mg-independent, high Km phosphodiesterase increased 20-fold throughout the 108 h cultivation . The specific activity of the Mg-dependent, low Km phosphodiesterase about doubled during glucose utilization and fell back to the initial level as the cells entered stationary phase.

Eur J Biochem, 1984 Dec 3, 145(2), 365 - 71
Glucose-6-phosphate dehydrogenase . A transferred nuclear Overhauser enhancement study of NADP+ conformations in enzyme-coenzyme binary complexes; Gronenborn AM et al.; The conformation of NADP+ in glucose-6-phosphate-dehydrogenase--NADP+ binary complexes has been investigated using proton-proton transferred nuclear Overhauser enhancement measurements to determine interproton distance ratios between bound NADP+ protons . The enzymes from Saccharomyces cerevisiae (brewer's yeast and baker's yeast) and Hansenula jadinii (Candida utilis, Torula utilis) form binary complexes with NADP+ in which the glycosidic bond of the adenine moiety is in the anti conformation whereas that of the nicotinamide moiety exists as a syn (69-70%)/anti (30-40%) mixture . The enzymes have similar subunit sizes (Mr approximately 58 000) and it is shown that they bind NADP+ in essentially similar conformations . Inactivation of the baker's yeast enzyme with acetylsalicylic acid caused little if any alteration in the conformation of bound NADP+, and the presence of NADP+ during inactivation afforded very little protection to the enzyme . Inactivation rates were, however, lower in the presence of glucose 6-phosphate . It is concluded that the epsilon-amino group of the lysine residue that is acetylated during the inactivation reaction with acetylsalicylic acid is not necessary for binary complex formation between the enzyme and NADP+, but that it is situated in a part of the molecule affected by formation of the enzyme--glucose-6-phosphate complex . The implication of the findings for the catalytic process, and related evolutionary aspects, are discussed briefly.

Onderstepoort J Vet Res, 1984 Dec, 51(4), 245 - 8
Preparation and properties of a crude extract toxic to guinea-pigs from Pachystigma pygmaeum, a plant causing heart failure in ruminants; Verschoor JA et al.; Pachystigma pygmaeum is a rubiaceous plant which causes heart failure after ingestion by ruminants . Isolation or characterization of a toxic entity has hitherto been unsuccessful . Extraction of the plant material with water or organic solvents gave poor yields of toxicity in guinea-pigs because of some unusual properties of the toxic entity . We present a method whereby liberation of a toxic entity from the plant material was achieved by fermentation with baker's yeast to give a crude extract with sufficient yield of toxicity for use in further isolation work.

Biochem Biophys Res Commun, 1984 Nov 14, 124(3), 807 - 14
Coupling between redox and acid-base energy by cytochrome b-564 in Baker's yeast mitochondria; Hervas M et al.; Baker's yeast mitochondrial cytochrome b-564 is characterized by exhibiting both a labile pH-independent high-potential form (E'o, pH 7 = + 190 mV) and a stable pH-dependent (pKa = 6.8) low-potential form (E'o, pH 7 = + 70 mV) . The different behavior of these two forms of cytochrome b-564 versus pH seems to be a decisive factor for transduction of redox energy into acid-base energy in oxidative phosphorylation site 2 . Deenergizing treatments, such as ADP plus Pi, result in the conversion of all the mitochondrial cytochrome b-564 into its low-potential form, whereas energization with ATP specifically transforms the cytochrome into its high-potential form, the ATP effect being neutralized by the ATPase inhibitor oligomycin and by the uncoupler FCCP . Accordingly, a minimal model for coupling between redox energy and acid-base energy through an electronically energized and protonated ferricytochrome b-564 intermediate is proposed . The energy-transducing properties of mitochondrial cytochrome b-564 seems to be shared by chloroplast cytochrome b-559.

J Chromatogr, 1984 Oct 26, 303(1), 39 - 51
Parameters determining affinity partitioning of yeast enzymes using polymer-bound triazine dye ligands; Johansson G et al.; Triazine dyes, bound to polyethylene glycol, have been used to influence the partition of some enzymes within a dextran-polyethylene glycol-water two-phase system . The enzymes, present in a protein extract from baker's yeast, included glucose 6-phosphate dehydrogenase, glyceraldehyde phosphate dehydrogenase, 3-phospho-glycerate kinase and alcohol dehydrogenase . The partition coefficients of the enzymes could be changed by a factor of 10-500 in favour of the polyethylene glycol-rich phase, while the partition of bulk protein was much less affected . The influence of the concentration of polymer-bound dye and phase-forming polymers, temperature, pH, kind and concentration of salt and the presence of nucleotides on this affinity partitioning effect was studied . The extraction was effective even at high concentrations of dye and protein (40 g/l) . A partial purification (32-fold) of glucose 6-phosphate dehydrogenase was carried out by an extraction in five steps.

Eur J Biochem, 1984 Oct 15, 144(2), 345 - 51
Baker's yeast flavocytochrome b2 . A mechanistic study of the dehydrohalogenation reaction; Urban P et al.; It has been shown that reduced flavocytochrome b2 not only catalyzes reduction of bromopyruvate {P . Urban, P.M . Alliel and F . Lederer (1983) Eur . J . Biochem . 134, 275-281} but also transforms it into pyruvate in a reductive elimination process . The dehydrohalogenation reaction also takes place when oxidized enzyme acts on bromolactate, but the reaction is more difficult to observe under these conditions because of its low efficiency compared to the normal oxidative process . The maximal rates of pyruvate production from bromopyruvate and chloropyruvate differ by a factor of less than 10, whereas elimination from fluoropyruvate cannot be detected . These results support a mechanism in which the dehydrohalogenation reaction takes place from a carbanion intermediate of the normal reductive-oxidative pathway.

Biochem Int, 1984 Oct, 9(4), 455 - 61
Chromatographic differentiation of the mitochondrial and cytosolic fumarases of rat liver and Baker's yeast and differential induction of two fumarases of Baker's yeast; Hiraga K et al.; The mitochondrial and cytosolic fumarases of rat liver could be separated from each other by Bio-Gel HTP column chromatography, showing that there are some conformational differences between the native proteins of the mitochondrial and cytosolic fumarases which have been indistinguishable by their physicochemical, catalytic, and immunochemical properties . The fumarase associated with the mitochondrial fraction of Baker's yeast could also be separated from that located in the cytosol by the Bio-Gel HTP column chromatography . By applying this chromatography to the differential determination of the fumarase activities in the Baker's yeast cultured under various conditions, it was shown that the mitochondrial and cytosolic fumarases in the cell are regulated by independent control systems.

Vopr Pitan, 1984 Sep-Oct, (5), 59 - 62
{Effect of parenterally administered baker's yeast autolysates on normalization of the plasma aminogram in acute hepatitis in the rabbit}; Nekliudov AD et al.; A study was made of the effect of autolysine--a mixture of amino acids and lowest peptides, obtained by autolysis of baker yeast biomass, on the normalization of blood plasma and brain tissue aminogram in rabbits with acute hepatitis caused by D-galactosamine . 3,6% isoleucine, leucine and valine as well as 0,9% sodium chloride served as control . Parenteral administration of autolysine in an amount of 4 g conventional protein per kg bw a day entailed a substantial normalization of the amino acid spectrum of blood plasma and brain.

Mol Cell Biochem, 1984 Sep, 64(1), 39 - 44
Multiforms of megamodulin-dependent protein kinases from baker's yeast; Kuo WN; Multiforms of megamodulin-dependent protein kinases (M-PK) were partially purified from baker's yeast by excluding endogenous megamodulin with histone, and then by gel filtration with Sephadex G-200 . The stimulation of M-PK in the presence of Mg2+, Mn2+ or Co2+ was enhanced by yeast megamodulin . In addition, similar augmented activity of M-PK was also noted in the presence of Mg2+ by megamodulins prepared from E . coli, bovine brain and wheat germ.

Anal Biochem, 1984 Aug 15, 141(1), 79 - 82
The purification of yeast glucose 6-phosphate dehydrogenase by dye-ligand chromatography; Farmer EE et al.; Glucose 6-phosphate dehydrogenase (EC 1.1.1.39) has been purified to homogeneity from baker's yeast by a simple procedure involving affinity elution from a column of red triazine dye, H-8BN, immobilized to Sepharose 6B . Eight milligrams of homogeneous protein is obtained in 53% yield from 200 g of dried yeast . This represents the first published purification of the enzyme from Saccharomyces Cerevisiae.

Eur J Biochem, 1984 Jul 16, 142(2), 287 - 9
The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria; Tomasselli AG et al.; The diastereomers of adenosine 5'-O-{1-thio}triphosphate (ATP{alpha S}) and adenosine 5'-O-{2-thio}triphosphate (ATP{beta S}) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2) . Similarly, the diastereomers of guanosine 5'-O-{thio}triphosphate (GTP{alpha S}) and guanosine 5'-O-{2-thio}triphosphate (GTP{beta S}) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3) . Furthermore the diastereomers of guanosine 5'-O-{1-thio}diphosphate (GDP-{alpha S}) have been used to assign Mg2+ coordination to GDP when bound to AK3 . The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) {Kalbitzer et al . (1983) Eur . J . Biochem . 133, 221-227} and from baker's yeast (AKy) {Tomasselli and Noda (1983) Eur . J . Biochem . 132, 109-115} . In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP {beta S} or GTP {beta S} respectively on changing the metal ion . In contrast, there is no reversal of specificity for the diastereomers of ATP {alpha S} or GTP{alpha S}, or for GDP{alpha S} in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group . The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.

Biochem Int, 1984 Jul, 9(1), 9 - 16
The carboxyl group in the active center of transketolase; Meshalkina LE et al.; Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K . In both cases the kinetics of inactivation is biphasic, which agrees with the presence of two active centers in the enzyme molecule differing in their sensitivity to the inhibitors . There is some evidence that inactivation of transketolase is due to modification of carboxyl groups of enzyme . Complete inactivation is achieved by modification of one carboxyl per active site of the enzyme . The experimental results suggest that the carboxyl group is essential for the enzymatic activity of transketolase.

J Biochem (Tokyo), 1984 Jun, 95(6), 1551 - 7
Properties of calcium-dependent regulatory proteins from fungi and yeast; Nakamura T et al.; Calmodulins were isolated from vegetative mycelia of Basidiomycetes fungi, Agaricus campestris and Coprinus lagopus . These calmodulins showed similar mobilities to those of animal calmodulins on nondenaturing polyacrylamide gel electrophoresis in the presence or absence of Ca2+ . The molecular weights of both calmodulins were determined to be 16,000 . Agaricus calmodulin consisted of 148 amino acids including epsilon-N-trimethyllysine and cysteine . The UV-absorption spectrum showed the relatively high content of phenylalanine in Basidiomycetes calmodulins . The difference UV-absorption spectrum due to the blue shift by Ca2+ was observed . Both calmodulins activated muscle myosin light chain kinase and pea NAD+ kinase in a Ca2+-dependent manner, and the activities were inhibited by trifluoperazine or chlorpromazine . A calmodulin-like protein was partially purified from baker's yeast, Saccharomyces cerevisiae . However, detection of a calmodulin-like protein in prokaryotes was not successful.

Eur J Biochem, 1984 May 2, 140(3), 531 - 7
NAD{S}, an NAD analogue with reduced susceptibility to phosphodiesterase . Chemical synthesis and enzymic properties; Meyer T et al.; The chemical synthesis of adenosine(5') {alpha-thio}diphospho(5')ribofuranosyl-nicotinamide (NAD{S}) is described . The product occurs as a pair of diastereomers with different configuration at the sulfur-bearing phosphorus atom . The diastereomers were separated by high-performance liquid chromatography and their absolute configuration was determined after chemical degradation to the ADP{alpha S} diastereomers and chromatographic comparison with enzymically synthesized ADP{alpha S} diastereomers of known absolute configuration . Additional support for this assignment is based on different rates in the phosphodiesterase-catalyzed hydrolysis . Furthermore the synthesis of {14C}NAD{S} is described . The coenzyme activity of NAD{S} in the reaction with alcohol dehydrogenase from baker's yeast and lactate dehydrogenase from pig heart is very similar to that of beta-NAD . Also, NAD and NAD{S} serve equally well as substrates for NAD glycohydrolase from calf spleen . In contrast, no reaction was detected with NAD pyrophosphorylase, and hydrolysis of the separated NAD{S} diastereomers with snake venom phosphodiesterase showed a 26-fold and a 33-fold slower reaction rate than that of NAD . Nucleotide pyrophosphatase was less sensitive to the S substitution, hydrolyzing NAD{S} 14-times slower than NAD . Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cell nuclei accepted NAD{S} as a substrate but the reaction was significantly slower and approached saturation at much lower values than with NAD . Alkaline hydrolysis of the products insoluble in trichloroacetic acid yielded AMP{S} as the main derivative . It is concluded that with NAD{S} as a substrate the nuclear acceptors were nearly exclusively mono(ADP-ribosyl) ated .

Biochem Biophys Res Commun, 1984 Apr 30, 120(2), 467 - 73
A re-evaluation of the molecular weight of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase; Rittenhouse J et al.; In contrast with previous results that indicate that Saccharomyces cerevisiae fructose-1,6-bisphosphatase is a dimer of 56,000 molecular weight subunits, we find that the subunit Mr of the enzyme purified from baker's yeast is 40,000 . The same subunit Mr was observed in immunoprecipitates of crude supernatants of baker's yeast and S . cerevisiae cultures, as well as in acid-extracts of cells detected by immunoblotting, suggesting that the native subunit indeed has a Mr of 40,000 and it has not been produced from a larger polypeptide . Complete immunoprecipitation of fructose-1,6-bisphosphatase activity with saturating concentrations of specific antibody suggests that there is only one fructose-1,6-bisphosphatase isozyme in S . cerevisiae . The Mr of the purified enzyme determined by size exclusion HPLC suggests that it has a tetrameric structure characteristic of fructose-1,6-bisphosphatases from a broad phylogenetic spectrum.

Eur J Biochem, 1984 Apr 16, 140(2), 281 - 7
Deoxyribonucleotide biosynthesis in yeast (Saccharomyces cerevisiae) . A ribonucleotide reductase system of sufficient activity for DNA synthesis; Lammers M et al.; Ribonucleotide reductase, the central enzyme of DNA precursor biosynthesis, has been isolated and characterized from baker's yeast . The enzyme activity, measured in extracts from three different, exponentially growing yeast strains, is high enough to meet the substrate requirement of DNA replication, in contrast to very low activities found in most other organisms . In thymidylate-permeable yeast cells ribonucleotide reductase activity is stimulated under both starvation and excess of intracellular dTMP . On the other hand growth of yeast in presence of 20 mM hydroxyurea did not increase enzyme activity . Yeast ribonucleotide reductase is composed of two non-identical subunits, inactive separately, of which one binds to immobilized dATP . The relative molecular mass of the holoenzyme is about 250 000 . The enzyme reduces all four natural ribonucleoside diphosphates with comparable efficacy . GDP reduction requires dTTP as effector, ADP reduction is stimulated by dGTP, whereas pyrimidine nucleotide reduction is stimulated by any deoxyribonucleotide and ATP . Enzyme activity is independent of exogenous metal ions and is insensitive towards chelating agents . Hydroxyurea inactivates yeast ribonucleotide reductase in a slow reaction; half-inhibition (I50) is reached only at 2-6 mM hydroxyurea concentration . Up to 50% reactivation occurs spontaneously after removal of the inhibitor . In accord with previous attempts by others, extensive purification of the yeast enzyme has failed owing to its extreme instability in solution; the half-life of about 11 h could not be influenced by any protective measure . Taken together, yeast ribonucleotide reductase combines features known from Escherichia coli and mammalian enzymes with differing, individual properties.

Biochem J, 1984 Apr 15, 219(2), 511 - 8
Characterization of two trehalases in baker's yeast; Londesborough J et al.; Trehalase activities at pH 5 (not inhibited by EDTA) and pH 7 (inhibited by EDTA) were present in the soluble fraction of disintegrated commercial baker's yeast . The pH 5 activity binds strongly to concanavalin A, is only partially salted out by saturated (NH4)2SO4, has an apparent Mr of 215000 (by gel filtration) and is an acidic protein . It has a Km of 1.4 mM, a broad pH optimum (at 40 mM-trehalose) between pH 4 and 5, and is activated by about 30% by 20-300 mM neutral salts such as KCl, NaNO3 and MnCl2 . The enzyme is strongly inhibited by acetic acid/acetate buffers, with a Ki of about 15 mM-acetic acid . The pH 7 activity does not bind to concanavalin A, is salted out at 20-32% (w/v) (NH4)2SO4 and has an Mr of 170000 (by gel filtration) . It is absolutely dependent on Ca2+ or Mn2+ ions (Mg2+ is ineffective) and strongly inhibited by neutral salts in the 20-100 mM range . It can be activated by treatment with MgATP in the presence of cyclic AMP . Activation decreases, but does not abolish, the Ca2+ requirement, and does not change the Km for trehalose (5.7 mM) or shift the sharp pH optimum at pH 6.7 (at 40 mM-trehalose).

Biokhimiia, 1984 Apr, 49(4), 607 - 10
{Incorporation of yeast tRNA into mouse L 1210 lympholeukemic cells}; Drize NI et al.; {32P}tRNA from baker's yeast is incorporated without degradation into lympholeukotic cells of L1210 mice . The tRNA incorporation determined after tRNA hydrolysis on cell surface by RNAase increases linearly with a rise in the initial concentration from 0.5 to 500 micrograms per ml . According to gel electrophoresis of intracellular nucleic acids, after a 3 hour incubation the {32P}tRNA incorporated into the cells by 50% to form tRNA fragments without any conspicuous reutilization . The kinetic curve of tRNA incorporation during the first 60 min demonstrates a severalfold decrease in the initial maximal incorporation of {32P}tRNA into the cells (2 min), with a subsequent restoration of the incorporation within 2-3 hours.

Proc Natl Sci Counc Repub China B, 1984 Apr, 8(2), 124 - 8
Enzymatic preparation of seasoning 5'-nucleotides from baker's yeast; Chen HJ et al.; Chemical phosphorylation with sodium trimetaphosphate (STMP) as a modifying agent was used to prepare functional protein isolate and dissociated nucleic acid (mostly RNA) from baker's yeast . The majority of protein-RNA complex in the disintegrated yeast cells was first extracted with an aqueous alkaline solution (pH 12, 40 degrees C) followed by phosphorylation with STMP under the same condition for 6 hrs . An apparent dissociation of protein-RNA complex occurred due to the covalent attachment of anionic phosphate groups onto yeast protein molecules . The nucleic acid residued in the supernatant after removal of modified protein isolate by isoelectric precipitation was recovered by reprecipitating at pH 2 followed by converting it to 5'-nucleotides with malt rootlets 5'-phosphodiesterase as well as red marine algal adenylate deaminase . This coherent process provided a preparation of food-usable functional protein isolate and 5'-nucleotides from baker's yeast.

Z Naturforsch {C}, 1984 Mar-Apr, 39(3-4), 276 - 81
Aurintricarboxylic acid and polynucleotides as novel inhibitors of ribonucleotide reductases; Baumann H et al.; Ribonucleoside diphosphate reductases isolated from Escherichia coli, baker's yeast, Ehrlich ascites tumor cells, and unicellular green alga (Scenedesmus obliquus) are inhibited strongly and uniformly by the polymeric triphenylmethane dye, aurintricarboxylic acid . The molecule appears to interact simultaneously with the enzyme's various nucleotide and catalytic (iron-organic radical) sites . Oligo- and polyribonucleotides are also inhibitory . These reactions serve as models of the probably physiologic regulation of ribonucleotide reduction exerted by natural inhibitors . Partial characterization of an inhibitor fraction found in wheat seed embryo is described.

Eur J Biochem, 1984 Feb 15, 139(1), 59 - 74
Primary structure of flavocytochrome b2 from baker's yeast . Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides; Ghrir R et al.; Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha . Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues) . The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns . The various systems tested are presented and compared . The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described . The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary . Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide . Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography . Peptides were sequenced mostly in the liquid phase sequenator . The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work . Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region {Guiard, B . and Lederer, F . (1976) Biochimie (Paris) 58, 305--316; Ghrir, R . and Lederer, F . (1981) Eur . J . Biochem . 120, 279--287}, the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides . Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity . Structural predictions indicate an alteration of alpha helices and beta structure . The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.

Acta Microbiol Hung, 1984, 31(3), 197 - 206
Synergistic interaction of baker's yeast and iron in the enhancement of bacterial virulence; Joo I et al.; The virulence-enhancing interaction of baker's yeast and different iron preparations (ferric ammonium citrate and iron dextran) was tested in mice challenged with Salmonella typhi and Vibrio cholerae (Inaba and Ogawa) strains . The virulence-enhancing effect of the yeast + iron combination increased significantly as compared to that of either yeast or iron alone . Toxicity assays of the single and combined baker's yeast and iron preparations by the mouse weight gain test have shown that the combinations are considerably more toxic than either single agent, probably owing to the presence of yeast . Examination of the single and combined preparations for influence on body temperature of mice has revealed a general hypothermic action, which was strongest in the combinations, owing again to the yeast . Theoretical considerations on the underlying mechanism of the virulence-enhancing effect have supported the hypothesis that the effect might be associated with the strong hypothermic action produced by baker's yeast and baker's yeast + iron combinations, in as much as hypothermia increases the production of siderophores which ensure the acquisition of iron indispensable for bacterial growth.

Enzyme, 1984, 31(3), 176 - 80
D-glucose anomeric preference of hexokinases from animals and yeast; Okuda J et al.; The D-glucose anomeric preference of hexokinases partially purified from animals (rat, mouse, and chicken) and baker's yeast (Saccharomyces cerevisiae) were investigated by the assay system with glucose-6-phosphate dehydrogenase as a coupling enzyme . With low Km hexokinases in animal tissues and cells, the ratios of Vmax for the beta-anomer to Vmax for the alpha-anomer (V beta/V alpha) were within a range from 1.3 to 1.5 . In yeast, the V beta/V alpha value was 1.1 for hexokinase A, 0.8 for hexokinase B, and 1.4 for glucokinase . The possible explanation for D-glucose anomeric preference of hexokinase is discussed.

J Biochem (Tokyo), 1984 Jan, 95(1), 137 - 45
Purification and properties of a membrane-bound phospholipase B from baker's yeast (Saccharomyces cerevisiae); Ichimasa M et al.; Phospholipase B bound tightly to the membrane fraction of baker's yeast (Saccharomyces cerevisiae) was purified approximately 226-fold by extraction with sodium deoxycholate (DOC), acetone precipitation, ammonium sulfate fractionation, and column chromatographies on Phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Sepharose 4B . Only one major activity peak with an apparent molecular weight of 330,000 was detected by gel filtration on Sepharose 4B . However, two glycoprotein bands, one major and one minor, were evident on SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol . Isoelectric focusing also revealed two activities, pI 3.4 (major) and pI 3.0 (minor) . The purified enzyme had phospholipase B activity (in the presence of DOC) and lysophospholipase activity in a ratio of 1:4.8 . Both activities had an optimum pH of 3.5-4.0 . Phospholipase B activity was appreciably stimulated only by DOC among bile acids, but lysophospholipase activity was markedly inhibited by them . Both activities were not stimulated by Ca2+ and inhibited by SDS, Triton X-100, Fe3+ and Al3+ . The purified enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups . It preferentially hydrolyzed 1-acyl-sn-glycero-3-phosphocholine and, to a lesser degree, 2-acyl-sn-glycero-3-phosphocholine.

Biochem Biophys Res Commun, 1983 Nov 30, 117(1), 259 - 67
Large scale purification and structural properties of yeast aspartyl-tRNA synthetase; Lorber B et al.; A large scale purification procedure of baker's yeast aspartyl-tRNA synthetase is described which yields more than 200 mg pure protein starting from 30 Kg of wet commercial cells . The synthetase is an alpha 2 dimer of Mr = 125,000 +/- 5,000 which can be crystallized (J . Mol . Biol . 138, 1980, 129-135) . The enzyme has an elongated shape with a Stokes radius of 50 A and a frictional ratio of 1.5 . The synthetase has a tendency to aggregate but methods are described where this effect is overcome.

FEBS Lett, 1983 Nov 14, 163(2), 175 - 80
Glycosylation of yeast aspartyl-tRNA synthetase . Affinity labelling by glucose and glucose 6-phosphate; Colas B et al.; Several lines of evidence establish that the crystallizable aspartyl-tRNA synthetase from Baker's yeast contains some covalently bound glucose: (i) a positive staining of the enzyme was obtained after polyacrylamide gel electrophoresis followed by the concanavalin A-peroxidase test which is specific for glucose and mannose containing proteins; (ii) thin-layer chromatography and gas-liquid chromatography revealed the presence of glucose in enzyme hydrolysates; (iii) immunoaffinoelectrophoresis in agarose gels containing concanavalin A and antibodies raised against aspartyl-tRNA synthetase showed that the enzyme was able to precipitate entirely in the lectin . Finally incubation of the enzyme with {14C}glucose or {14C}glucose 6-phosphate led to the incorporation of radioactivity into trichloroacetic acid-precipitable protein . Indeed immunoprecipitation of {14C}glucose-labelled aspartyl-tRNA synthetase with specific antibodies using the rocket method followed by autoradiography gave a radioactive peak . This last result also demonstrates the possibility of in vitro glycosylation of yeast aspartyl-tRNA synthetase.

Z Naturforsch {C}, 1983 Nov-Dec, 38(11-12), 1069 - 71
Antibodies against the alpha-factor pheromone of Saccharomyces cerevisiae; Tillmann U et al.; The mating pheromone of baker's yeast, the alpha-factor, is a dodeka-/tridecapeptide, which is not antigenic by itself . It was coupled to succinylated thyroglobulin by the carbodiimide procedure to facilitate selective coupling of the alpha-factor mainly by its N-terminal region . Antibodies against this conjugate were raised in rabbits . After selective precipitation of the rabbit antiserum with succinylated carrier prior to the radial double diffusion test (Ouchterlony) specific antibodies against the coupled alpha-factor could be detected.

Anal Biochem, 1983 Oct 1, 134(1), 210 - 5
Purification and fluorometric assay of proteinase A from yeast; Yokosawa H et al.; Proteinase A was purified from commercial baker's yeast to homogeneity by using affinity chromatography . Simple and sensitive fluorometric assay procedures were developed for this enzyme, where Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide and dimethylcasein were used as synthetic substrate and protein substrate, respectively . Proteinase A cleaved the former substrate at the Leu-Val bond . The extent of the cleavage was monitored by measuring the amount of fluorescent 7-amino-4-methylcoumarine produced by the successive cleavage with an auxiliary enzyme, aminopeptidase M, or by measuring the fluorescence generated by the reaction of newly formed amino groups with fluorescamine . In the assay with the latter substrate, the amounts of newly formed amino groups were measured by the reaction with fluorescamine . The optimal pH of the reaction was 5.0 to 5.5 . The methods were applied to the study of the effects of denaturing agents on the activity of proteinase A.

Arch Biochem Biophys, 1983 Oct 1, 226(1), 19 - 26
Effect of polyamines on palmitoyl-coenzyme A-caused inhibition of glucose-6-phosphate dehydrogenase from baker's yeast; Mita M et al.; Aliphatic polyamines reversed the inhibition of baker's yeast glucose-6-phosphate dehydrogenase by palmitoyl-CoA . The reversal effects of the polyamines on the inhibition were related not only to the number of amino (imino) groups but also to the carbon chain length of polyamine . The relieving constant (Kr) was calculated to determine the absolute concentration of each examined polyamine required to relieve the enzyme from the inhibition . A smaller Kr value indicates a stronger relieving activity . An ethylamine derivative with a large number of amino groups was more effective in reversing the palmitoyl-CoA inhibition . The Kr values of long-chain aliphatic diamines, triamines, and tetramines were smaller than those of short-chain ones . However, n-butylamine and ornithine, composed of four carbons like putrescine, had markedly larger Kr values than putrescine . Substitution of one or two amino groups by carboxyl or hydroxyl groups also appeared to relieve the enzyme from the inhibition . These results suggest that the reversal effect on palmitoyl-CoA inhibition is one of the important roles of polyamines . Furthermore, it is possible that the interaction of palmitoyl-CoA and polyamines physiologically regulates the pentose monophosphate cycle in baker's yeast.

Prikl Biokhim Mikrobiol, 1983 Sep-Oct, 19(5), 692 - 5
{Amino acid analysis of culture media and autolysates by capillary gas chromatography}; Vitt SV et al.; N-heptofluorbutyryl-O-propyl ethers from 20 natural amino acids were analysed on a capillary column Am-Ac by gas chromatography . A high reproducibility of the quantitative estimation of the compounds studied was achieved not only for the standard mixtures but when analysing the amino acid composition of the autolysates of the baker's yeast and cultural media.

Arch Biochem Biophys, 1983 Sep, 225(2), 667 - 78
Purification and properties of phosphorylase from baker's yeast; Becker JU et al.; A rapid, reliable method for purification of phosphorylase, yielding 200-400 mg pure phosphorylase from 8 kg of pressed baker's yeast, is described . The enzyme is free of phosphorylase kinase activity but contains traces of phosphorylase phosphatase activity . Phosphorylase constitutes 0.5-0.8% of soluble protein in various strains of yeast assayed immunochemically . The subunit molecular weight (Mr) of yeast phosphorylase is around 100,000 . The enzyme is composed of two subunits in various ratios, differing slightly in molecular weight and N-terminal sequence . Both are active . Only the enzyme species containing the larger subunit can form tetramers and higher oligomers . The activated enzyme is dimeric . Correlated with specific activity (1 to 110 U/mg), phosphorylase contained between less than 0.1 to 0.74 covalently bound phosphate per subunit . Inactive forms of phosphorylase could be activated by phosphorylase kinase and {gamma-32P}ATP with concomitant phosphorylation of a single threonine residue in the aminoterminal region of the large subunit . The small subunit was not labeled . The incorporated phosphate could be removed by yeast phosphorylase phosphatase, resulting in loss of activity of phosphorylase, which could be restored by ATP and phosphorylase kinase.

J Biol Chem, 1983 Aug 25, 258(16), 9745 - 54
Kinetic analysis of nicotinate phosphoribosyltransferase from yeast using high pressure liquid chromatography; Hanna LS et al.; A new procedure has been designed for the purification of nicotinate phosphoribosyltransferase and orotate phosphoribosyltransferase from the same baker's yeast extract . Using purified nicotinate phosphoribosyltransferase, the enzyme-catalyzed formation of nicotinate mononucleotide was analyzed using a new high pressure liquid chromatographic assay (Hanna, L., and Sloan, D . L . (1980) Anal . Biochem . 103, 230-234) . Initial velocity measurements and product inhibition studies, with pyrophosphate, were performed . In addition, this assay procedure was used to demonstrate that purified nicotinate phosphoribosyltransferase possesses an ATPase activity in the presence of either product (pyrophosphate or nicotinate mononucleotide (NaMN} but in the absence of 5-phosphoribosyl alpha-1-pyrophosphate (P-Rib-PP) . Moreover, exchanges of radioactivity between specific substrate/product pairs {( 14C}nicotinate/NaMN and {32P}PPi/P-Rib-PP) in the absence of other substrates were not observed when these pairs were incubated with nicotinate phosphoribosyltransferase, and binding of {14C} nicotinate to nicotinate phosphoribosyltransferase was not detected in the presence of ATP . In contrast, an exchange of label between ATP and {14C}ADP was characterized in the absence of other substrates and in the presence of either P-Rib-PP or PPi . These results indicate that nicotinate phosphoribosyltransferase proceeds through the use of an ordered Uni Uni Bi Ter Ping Pong kinetic mechanism during which ATP reacts with nicotinate phosphoribosyltransferase to form ADP and a previously described phosphorylated enzyme (Kosaka, A., Spivey, H . O., and Gholson, R . K . (1977) Arch . Biochem . Biophys . 179, 334-341) . Thereafter, P-Rib-PP and nicotinate bind in order to the active site, to produce PPi and NaMN which are released in a random order followed by Pi . The Km values for ATP, P-Rib-PP, and nicotinate were calculated to be 70 +/- 10, 24 +/- 3, and 23 +/- 4 microM, respectively, whereas a value for Ki(PRPP) of 5 +/- 1 microM was determined.

FEBS Lett, 1983 Aug 22, 160(1-2), 21 - 4
Isolation of deuterated histones from yeast grown on media dissolved in 2H2O; Hamana K et al.; We have succeeded in growing Saccharomyces cerevisiae (baker's yeast) on media containing 2H2O and isolating the core histones highly deuterated in the non-exchangeable positions . The deuterated histones obtained here are of great value for their possible widespread use for structural studies of chromatin.

Eur J Biochem, 1983 Aug 1, 134(2), 275 - 81
On the transhydrogenase activity of baker's yeast flavocytochrome b2; Urban P et al.; It is shown that, when baker's yeast flavocytochrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate . The heme remains reduced during the reaction . It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates . Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control . Determinations of the steady-state deuterium isotope effect show that the rate-limiting step in the oxidation of halogenolactates is abstraction of the alpha-hydrogen (probably as a proton), as is the case for lactate itself . According to the principle of microscopic reversibility, the rate-limiting step in the reverse reaction must be protonation of the putative carbanion.

J Immunol Methods, 1983 Jul 29, 61(3), 359 - 65
A simple and rapid flow cytometric method for routine assessment of baker's yeast uptake by human polymorphonuclear leukocytes; Derer M et al.; A new method for measuring uptake of baker's yeast (BY) by human polymorphonuclear leukocytes (PMN) using flow cytometry is described . The method correlates excellently with the visual method, is reproducible and provides a means for investigating the early phases of the phagocytic process as well as the phagocytic capacity of PMN . This quick and accurate method allows the counting of large numbers of cells, and monitoring of the process of particle uptake and has a considerable potential in the routine assessment of polymorph function in various clinical situations.

Biochem Biophys Res Commun, 1983 Jul 18, 114(1), 403 - 9
Stimulation of mammalian and yeast phosphoprotein phosphatases by megamodulins from various sources; Kuo WN; Phosphoprotein phosphatases (PPase) were isolated from either the rabbit cerebral cortex or Baker's yeast by excluding endogenous megamodulin with histone, and then by desalting cations with Bio-Gel P-6DG filtration . The stimulation of PPase in the presence of Mn2+ was greatly enhanced by megamodulins prepared from various sources including rabbit brain, Baker's yeast, wheat germ, and Escherichia coli (E . coli) . Moreover, the augmented activity of PPase was also demonstrated in the presence of {megamodulin-Mn2+} complex.

Biochimie, 1983 Jul, 65(7), 379 - 88
Primary structure of aspartyl-tRNA synthetase from baker's yeast: tryptic and CNBr peptides; Hounwanou N et al.; The cristallizable aspartyl-tRNA synthetase from Baker's yeast is a dimer made up of identical subunits (Mr 60,000) . We report here the results of tryptic digestion and cyanogen bromide cleavage which enabled us to align two lon stretches of sequence of 106 and 111 amino acids, respectively.

Biochim Biophys Acta, 1983 May 18, 744(3), 304 - 11
Improved purification and sulfhydryl analysis of thiosulfate reductase; Chauncey TR et al.; Thiosulfate reductase purified 900-fold from an extract of baker's yeast by a new procedure consisted of three charge-isomeric species, all with catalytic activity . Amino acid analysis of thiosulfate reductase and titrations with 5,5'-dithiobis(2-nitrobenzoate) and iodo{14C}acetate indicated only one cysteine residue per enzyme molecule . Alkylation of this cysteine with either iodoacetate or iodoacetamide did not inactivate the enzyme, indicating that sulfur-sulfur bond cleavage in the thiosulfate substrate does not require an enzymic sulfhydryl group as attacking nucleophile.

Biochem Int, 1983 May, 6(5), 673 - 83
Interaction of baker's yeast transketolase modified by 2,3-butanedione with anionic and nonanionic substrates; Usmanov RA et al.; Baker's yeast apotransketolase modified by 2,3-butanedione binds thiamine pyrophosphate, a coenzyme of transketolase, and forms a charge transfer complex which can be identified by the CD spectrum . The enzyme retains thereby its ability to bind anionic and nonanionic substrates . The affinity of the modified enzyme for donor substrates changes insignificantly, whereas Vmax decreases 5-10-fold . The results of the study show that, although the arginine residue of the active center is not involved in substrate binding, it is essential for catalysis.

Biokhimiia, 1983 May, 48(5), 772 - 81
{Function of the arginine residue in the active center of baker's yeast transketolase}; Usmanov RA et al.; The binding of anionic and non-anionic donor substrates to baker's yeast transketolase modified at the arginine residue by 2,3-butanedione was studied . The modified enzyme binds two thiamine pyrophosphate molecules per mole of protein, forms a complex with a charge transfer as recorded by circular dichroism (CD) spectra and retains its ability to bind anionic and non-anionic substrates . The values of the binding constants as determined from the CD spectra remain either unchanged or are changed very slightly . The values of the kinetic parameters, Km and V, for the transketolase-catalyzed oxidation reaction in the presence of anionic and non-anionic donor substrates were calculated . The Km values for both substrate groups were not affected by the enzyme modification, while those of V were decreased 5-10 times . The experimental data suggest that the arginine residue is not involved in the enzyme binding to the substrates but is catalytically essential.

Biochem J, 1983 May 1, 211(2), 515 - 7
Yeast copper-thionein can reconstitute the Japanese-lacquer-tree (Rhus vernicifera) laccase from the Type 2-copper-depleted enzyme via a direct copper(I)-transfer mechanism; Morpurgo L et al.; The Type 2-Cu-depleted laccase from the Japanese lacquer tree (Rhus vernicifera) can be reconstituted with CuSO4 aerobically and much more rapidly and efficiently under anaerobic reducing conditions . This is to be related to a more favourable conformation of a laccase in the reduced state, rather than to reduction of the metal ion . In fact, reconstitution with Cu(I)-thionein from baker's yeast (Saccharomyces cerevisiae) only proceeds under anaerobic reducing conditions, via a direct transfer of Cu(I).

Eur J Biochem, 1983 Apr 5, 131(3), 589 - 94
Affinity partitioning of phosphofructokinase from baker's yeast using polymer-bound Cibacron blue F3G-A; Johansson G et al.; 1 . Phosphofructokinase from baker's yeast is partitioned between the phases of an aqueous two-phase system, containing dextran (Mr = 500000) and poly(ethyleneglycol) (Mr = 6000), in favour of the dextran-rich phase . By covalent binding of the dye Cibacron blue F3G-A to poly(ethyleneglycol) the enzyme can be extracted to the phase rich in this polymer, i.e . affinity partitioning . 2 . The affinity partitioning effect, measured as the logarithmic increase of the partition coefficient by introducing polymer-bound Cibacron blue, depends on several factors . The influence of dye-polymer concentration, polymer concentration, polymer molecular weight, kind of salt and salt concentration, pH and temperature has been studied . 3 . The effect of ATP, ADP, AMP, ITP, fructose 1,6-bis-phosphate and fructose 6-phosphate show large differences in the binding strength of these substances to the Cibacron blue binding sites . AMP cannot compete with Cibacron blue while ATP is strongly competing . 4 . The use of affinity partitioning for enzyme isolation and determination of ligand binding is discussed, as well as possible mechanisms concerning this type of liquid/liquid extraction.

Biochem Int, 1983 Apr, 6(4), 443 - 50
Selective inhibition of an enzyme in crude cell extracts . The use of subunits modified with a half-of-the-sites reagent; Muronetz VI et al.; Yeast glyceraldehyde-3-phosphate dehydrogenase carboxymethylated at four active-site cysteine residues was incubated with a crude extract of baker's yeast . This resulted in a loss of the glyceraldehyde-3-phosphate dehydrogenase activity initially present in the extract . The extent of inactivation depended upon the ratio modified enzyme/enzyme present in the extract . Under appropriate conditions 63.1% inactivation of glyceraldehyde-3-phosphate dehydrogenase in crude extract could be achieved . The observed effect is explained in terms of hybridization between the carboxymethylated dimers of the purified enzyme and dimeric species of glyceraldehyde-3-phosphate dehydrogenase present in the crude extract, the inactivation being due to the influence of the half-of-the-sites reagent transmitted via the interdimeric contacts.

Mycopathologia, 1983 Mar 22, 81(3), 187 - 9
Influence of Fusarium and Myrothecium mycotoxins on dehydrogenase activity of Saccharomyces cerevisiae; Reiss J; The Fusarium and Myrothecium mycotoxins roridin A, diacetoxyscirpenol, verrucarin A, T-2 toxin and zearalenone (10(-2) and 10(-3) mg/ml) inhibit the unspecific dehydrogenase activity of baker's yeast (Saccharomyces cerevisiae) in vivo . The action of these toxins is in the same order as that of aflatoxin B1 . It is suggested that at least the trichothecenes decrease the dehydrogenase activity by an interaction with thiol groups of the active center of the enzymes.

Eur J Biochem, 1983 Mar 15, 131(2), 359 - 65
A cysteine cluster critical for flavin binding in flavocytochrome b2 from Baker's yeast; Pompon D et al.; We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome b2, a reaction which leads to the loss of flavin-binding capacity ('inactivation') {D . Pompon and F . Lederer (1982) Eur . J . Biochem . 129, 143-137} . For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit . In this work we report the results of sequence studies which elucidate the nature of the modification . The modified protein was cleaved with cyanogen bromide and the peptides separated on Sephadex G-100 and SP-Sephadex C-25 . 14C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting . It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion . Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule . Only two of the possible cross-links were found . These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme . These results, combined with those obtained in the affinity labeling reaction of holoenzyme with bromopyruvate {Alliel et al . (1982) Eur . J . Biochem . 122, 553-558}, show that the three residues are located in or close to the active site . Their possible role is discussed.

J Chromatogr, 1983 Mar 4, 257(2), 305 - 15
tRNA separation by high-performance liquid chromatography using an aggregate of ODS-Hypersil and trioctylmethylammonium chloride; Bischoff R et al.; High-performance liquid chromatography on a reversed-phase support treated with a tetraalkylammonium salt was used to separate tRNAs from baker's yeast . While resolution by this column appears to result from both anion-exchange and reversed-phase chromatography, it is the hydrophobic interactions which govern the separation of one tRNA from another . Chromatography of bulk tRNA resulted in a number of fractions with different amino acid acceptor activities and little cross-contamination . In some cases the column resolved several single nucleotide modifications of tRNAPhe . Using a 250 x 6.2 mm column it has been possible to chromatograph a minimum of 100 A260 units of tRNA without serious loss in resolution . tRNAs isolated from this column as the last step of a purification procedure have very high amino acid acceptor activities.

Eur J Biochem, 1983 Mar 1, 131(1), 65 - 80
Isoleucyl-tRNA synthetase from Baker's yeast . Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids; Freist W et al.; The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase has been investigated by initial rate kinetics . For aminoacylation of tRNAIle-C-C-3'dA with isoleucine, a bi-bi uni-uni ping-pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3'dATP; for aminoacylation with valine a bi-uni uni-bi ping-pong mechanism . For isoleucylation of tRNAIle-C-C-A(3'NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition . tRNAIle-C-C-2'dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P{NH}P as product analog and by isoleucinol . Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3'dATP and valinol . All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A . The Km values of the three substrates and the kcat values of the six reactions are given . For aminoacylation at the terminal 2'OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167 . For aminoacylation at the terminal 3'-OH group a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed . Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2'-OH group and conclusions drawn from experiments with modified tRNAs concerning 2',3'-specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNAs . The stability of Ile-tRNAIle-C-C-2'dA and Val-tRNAIle-C-C-2'dA, the lesser stability of Val-tRNAVal-C-C-2'dA and the instability of Thr-tRNAVal-C-C-2'dA are consistent with postulations for a 'pre-transfer' proofreading step for isoleucyl-tRNA synthetase and a 'post-transfer' hydrolytic editing step for valyl-tRNA synthetase at the terminal 3'OH group of the tRNA.

Int J Biochem, 1983, 15(11), 1295 - 304
Lipoxygenase from baker's yeast: purification and properties; Shechter G et al.; Lipoxygenase activity was extracted from the mitochondrial fraction of baker's yeast and was purified by affinity chromatography on a linoleyl aminoethyl sepharose column . Two lipoxygenases were eluted from the affinity column . The second enzyme eluted was characterized as a true lipoxygenase . The lipoxygenase eluted showed maximum activity at pH 6.5 with a Km of 2.68 X 10(-4) M on linoleate . The reaction products of the second lipoxygenase with linoleate were characterized by u.v., i.r., NMR spectra and mass spectrometry and were found to be: 9-hydroperoxy-octadeca-trans-10,cis-12-dienoic acid and 13-hydroperoxy-octadeca-cis-9,trans-11-dienoic acid.

Hoppe Seylers Z Physiol Chem, 1983 Jan, 364(1), 41 - 50
Lipoamide dehydrogenase from baker's yeast . Improved purification and some molecular, kinetic, and immunochemical properties; Heinrich P et al.; The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield . The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously) . The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex . The apoenzyme was reactivated when incubated with FAD but not FMN . As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors . Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD . NADH was a competitive inhibitor with respect to NAD (Ki 31 microM) . The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge . The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys . The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.

Microbios, 1983, 37(147), 15 - 21
Stimulatory protein kinase modulator, the cation-binding protein and megamodulin . 3 . Isolation from baker's yeast; Kuo WN et al.; Three peak-activity fractions of megamodulin were noted in Sephadex G-100 filtration of its crude preparation from baker's yeast (Saccharomyces cerevisiae) . Megamodulin thus prepared and its Mg2+-binding complex were shown not only to augment the activity of rabbit brain megamodulin-dependent protein kinase I (M-PK1) but also to interact with arginine-rich histone.

Mol Gen Genet, 1983, 190(2), 315 - 7
The cdc9 ligase joins completed replicons in baker's yeast; Johnston LH; The drug hydroxyurea has been found to affect the conditional DNA ligase mutant cdc9 in the same way as a wild type . Specific concentrations inhibit the joining of completed replicons leading to a substantial accumulation of these molecules . Upon removal of hydroxyurea and further incubation of cdc9 cells at the permissive temperature the replicons joined together, while in sharp contrast at the restrictive temperature no such joining occurred . However, a revertant of cdc9 able to grow at the restrictive temperature was also able to join replicons under these conditions, so the cdc9 ligase must be responsible for the assembly of completed replicons.

Biochem J, 1983 Jan 1, 209(1), 151 - 7
Purification and properties of tryptophan synthase from baker's yeast (Saccharomyces cerevisiae); Bailey CJ et al.; Tryptophan synthase was purified from baker's yeast . The purified enzyme exhibited one band on polyacrylamide-gel electrophoresis, had no detectable N-terminal amino acid and C-terminal alanine . The amino acid composition was close to that predicted by recent studies on the DNA sequence of the structural gene for the enzyme . Kinetic parameters for the following three activities were measured: indole-serine condensation, indolylglycerol phosphate lyase and the overall reaction of serine with 1-(indol-3-yl)glycerol 3-phosphate . The Km for indole was much lower than suggested by previous investigations, and the value of 11 microM was measured by a fluorimetric assay.

Enzyme, 1983, 29(2), 73 - 85
A kinetic study of the oxidation by molecular oxygen of the cytochrome chain of intact yeast cells, Acetobacter suboxydans cells, and of particulate suspensions of heart muscle; Ludwig GD et al.; The pre-steady state kinetics of the cytochrome c oxidase reaction with oxygen were studied by a variation in the reaction time between approximately 6 and 25 ms at oxygen concentrations less than 6 mumol/l . For baker's yeast, a pseudo-first-order velocity constant of approximately 150 s-1 at 1.3 mumol/l O2 was obtained corresponding to a second-order reaction between O2 and a3 at a forward velocity constant (k+1) of approximately 3 X 10(7) liter equiv.-1s-1 . Thus, the membrane-bound oxidase in the intact cell exhibits one of the most rapid enzyme-substrate reactions to be reported . The value is identical with that of Greenwood and Gibson on an isolated, solubilized cytochrome c oxidase . Similar values of k+1 are calculated from the turnover numbers {k+2 (a+2)} divided by the Km values (formula; see text) measured for these yeast preparations, which points to an almost negligible reverse reaction (k-1) compared to k+2(a+2) . Similar calculations for the membrane-bound cytochrome c oxidase of heart muscle give a value of k+1 approximately equal to 10(7) liter equiv.-1s-1 . The concordance of the different values of k+1 supports the view that the yeast cell wall does not impart a significant diffusion barrier to the transport of molecular oxygen . In contrast, Acetobacter suboxydans exhibits a much larger value for Km, and has a terminal oxidase of different kinetic parameters.

Int J Biochem, 1983, 15(6), 849 - 54
The flip-flop mechanism of the phosphorylation of yeast inorganic pyrophosphatase; Bakuleva NP et al.; 1 . An active monomeric form of inorganic pyrophosphatase from baker's yeast was prepared by maleylation of the protein at pH 10.5 . 2 . The dimeric and monomeric pyrophosphatase bound at non-catalytic sites 0.5 and 1.0 mol of slowly dissociating Pi per mol subunit, respectively . This stoichiometry was not affected on active site blockage with PPi . 3 . Added Pi accelerated the dissociation of Pi from the dimeric but not monomeric enzyme . 4 . Our results indicate a strong interaction to occur between the non-catalytic sites of two subunits of native pyrophosphatase which results in diminished stability of Pi binding to one of them.

Eur J Biochem, 1982 Dec, 129(1), 143 - 7
A residue critical for flavin binding in flavocytochrome b2 from Baker's yeast . Inactivation and labeling of flavin-free enzyme by 2-keto-3-butynoate; Pompon D et al.; The reagent 2-keto-3-butynoic acid is the product formed in the reaction between the suicide reagent 2-hydroxy-3-butynoate and a number of flavoproteins . We describe in this paper the inactivation of flavin-free flavocytochrome b2 by 2-keto-3-butynoate, in a rapid reaction which introduces 0.9 mol reagent for total inactivation . The modification results in loss of affinity for flavin and affects a cysteine residue . We also describe in this paper a simple enzymatic method for preparing 2-keto-3-butynoate, as well as some properties of the reagent, in particular its stability and susceptibility to nucleophilic attack . We show that at neutral pH it is highly specific for thiol compounds . Some properties of the adduct formed with glutathione are described . These experiments should pave the way for the use of 2-keto-3-butynoate with other proteins.

Gut, 1982 Dec, 23(12), 1037 - 43
Bacterial and fungal infection in children with fulminant hepatic failure: possible role of opsonisation and complement deficiency; Larcher VF et al.; Serious bacterial infection, including eight episodes of bacteraemia, developed in seven of 15 (47%) children with fulminant hepatic failure . Those with infections had a slightly higher leucocyte response than those who did not . Serum immunoglobulin concentrations were normal or raised in all patients . Opsonisation of heat-killed baker's yeast, functionally measured total haemolytic complement, C4, C5, total alternative pathway activity, factor B and D activity, and C3 concentrations were all significantly (p less than 0.005) reduced at presentation but returned to normal in those who survived . The severity of defects in yeast opsonisation, C4, and factor B activity at presentation were significantly correlated with the subsequent development of infection . In five patients bacteraemia occurred at a time when opsonisation and complement components were defective . Plasma infusions in vivo improved opsonisation in vitro and only one bacterial isolate was obtained within four days of such an infusion . Those patients who developed infection had received significantly (p less than 0.05) fewer plasma infusions than those who did not . Our findings suggest that both alternative and classical pathways of complement are defective in children with severe liver disease and may contribute to the susceptibility of such patients to infections . Plasma infusions might be useful in reducing the incidence of bacterial infection in such conditions.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7102 - 6
In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae; Mihara K et al.; We have isolated an outer mitochondrial membrane (OMM) fraction from baker's yeast . Saccharomyces cerevisiae, that possesses porin activity and contains a major polypeptide of 29,000 daltons . By analogy to similar data for an OMM fraction from rat liver and mung bean {Zalman, L . S., Nikaido, N . & Kagawa, Y . (1980) J . Biol . Chem . 255, 1771-1774}, the 29,000-dalton polypeptide of the isolated yeast OMM fraction has been tentatively identified as porin . Evidence to substantiate this identification was provided by the finding that both the porin activity and the 29,000-dalton polypeptide were entirely resistant when the OMM fraction was exposed to trypsin digestion, with the 29,000-dalton polypeptide being virtually the only polypeptide in the OMM fraction to be unaffected by trypsin digestion . There was no protection when trypsin digestion was carried out in the presence of detergent . Using monospecific antibodies, we have shown that yeast porin is apparently not synthesized as a larger precursor in a cell-free translation system . In vitro-synthesized porin could not be integrated into dog pancreas microsomal vesicles or into an isolated OMM fraction from yeast, either co- or posttranslationally . In vitro-synthesized porin, however, could be integrated posttranslationally into whole isolated mitochondria . This membrane specificity suggests that integration does not proceed by unassisted partitioning . The integration of porin into whole mitochondria occurred with fidelity by the criterion of its resistance to trypsin . Moreover, integration was not inhibited in the presence of the protonophore carbonyl cyanide m-chlorophenyl-hydrazone whereas translocation into the mitochondrial matrix of the in vitro-synthesized gamma subunit of F1-ATPase was inhibited.

Biochim Biophys Acta, 1982 Sep 15, 681(3), 484 - 95
Purification and characterization of highly purified cytochrome b from complex III of baker's yeast; T'sai AL et al.; A simple, high-yield purification procedure for cytochrome b from yeast Complex III has been developed . This procedure involves solubilization using chemical modification of the lysine residues with 3,4,5,6-tetrahydrophthalic anhydride followed by hydroxyapatite column chromatography . This purified cytochrome b has a heme content of 37.0 nmol cytochrome b/mg and a molecular weight on SDS gels of 25000-26000 . Amino acid analysis indicates high hydrophobicity and is very comparable to the composition deduced from the gene sequence (Nobrega, F.G . and Tzagoloff, A . (1980) J . Biol . Chem . 255, 9828-9837) . The latter data indicate a molecular weight of 42000 for the polypeptide; our heme analyses thus imply the presence of two hemes per polypeptide chain . Optical and MCD spectra are typical of a low-spin b-type cytochrome . MCD-potentiometric titration indicates a one-electron carrier with a single midpoint potential of -44 mV at pH 7.4 and 25 degrees C . The EPR spectrum of isolated cytochrome b has only one gz signal at 3.70, indicating that the 'strained' heme structure (Carter, K., T'sai, A . and Palmer, G . (1981) FEBS Lett . 132, 243--246) is still maintained . No indication of antimycin binding was demonstrated either by the direct-fluorescence method or binding-precipitation method although stoichiometric binding to the parent Complex III was readily demonstrated.

Biokhimiia, 1982 Sep, 47(9), 1483 - 7
{Interaction of inorganic pyrophosphatase from yeast with alkylated derivatives of Sepharose}; Plaksina EA et al.; The adsorption of inorganic pyrophosphatase from baker's yeast by alkylated polysaccharides involves both electrostatic and hydrophobic interactions . The enzyme is eluted by NaCl solutions of different ionic strengths depending on the adsorbent hydrophobicity . The degree of purification on different adsorbents varies from 2- to 80-fold.

Biochim Biophys Acta, 1982 Aug 23, 706(1), 141 - 3
Carboxypeptidase Y from Saccharomyces cerevisiae . Conformational differences reflected in kinetic behaviour in water and deuterium oxide; Nakagawa Y et al.; The glycoenzyme carboxypeptidase Y (peptidyl--amino-acid hydrolase, EC 3.4.16.1), from baker's yeast (British Fermentation Products Strain, Ng 72), of molecular weight 60 000, had a protein portion closely similar to those in the literature for carboxypeptidase Y isolations from other yeast sources, but was 25.3 wt% carbohydrate (mannose 83.3% by wt., glucosamine 10.3% by wt . with traces of galactose and galactosamine) . Circular dichroic spectra indicated that the enzyme lost its beta-structure as the pH was lowered from 8.08 to 4.16 . At p2H 8.22 in 2H2O media the conformation of this enzyme was different from that observed at pH 8.08 . A tyrosine residue appeared to be perturbed by lowering the pH of the medium . Carboxypeptidase Y was rapidly, and essentially irreversibly, inactivated at low p2H . The pH profile of kcat for the carboxypeptidase Y-catalysed hydrolysis of 4-nitrophenyltrimethylacetate showed two inflections at 45 degrees C: one at pKapp approximately 3.7 insensitive to temperature variation (ascribed to a carboxyl group), and one of pKapp approximately 5.7 markedly temperature-dependent and possibly caused by a histidine residue.

Biokhimiia, 1982 Aug, 47(8), 1289 - 92
{Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase}; Kasho VN et al.; The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E . coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared . For both phosphatases the type of interaction was found to be independent on the nature of substrate . For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively . In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction . The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.

Biokhimiia, 1982 Jul, 47(7), 1084 - 90
{Cooperative mechanism of phosphorylation of the monomeric and dimeric forms of inorganic pyrophosphatase from baker's yeast}; Bakuleva NP et al.; A comparative study of phosphorylation of native dimeric and artificial monomeric forms of inorganic pyrophosphatase and its fluoride-stabilized complex with PPi has been carried out . The maximal incorporation of Pi for the dimeric and monomeric proteins is 0.5 and 1 mole per mole of subunit, respectively . The saturation kinetic curves are suggestive of strong positive cooperative interactions . The value of the Hill coefficient (5.5) for the free dimeric enzyme drastically changes upon the active center blockage and/or transition to the monomeric enzyme . Acceleration of dephosphorylation induced by Pi in the presence of Mg2+ is observed only in the case of the dimeric protein . The data obtained indicate that phosphorylation of native dimeric pyrophosphatase occurs according to a "flip-flop" mechanism; the Pi binding in the active center exerts a strong influence on individual steps of the reaction.

Biochim Biophys Acta, 1982 Jun 14, 688(2), 295 - 304
Uptake and phosphorylation of 2-deoxy-D-glucose by wild-type and single-kinase strains of Saccharomyces cerevisiae; Franzusoff A et al.; The role of phosphorylation in sugar transport in baker's yeast was studied using 2-deoxy-D-glucose . In wild-type baker's yeast, 2-deoxy-D-glucose is accumulated as a mixture of the free sugar and several derivatives . Pool labeling experiments, designed to determine the temporal order of appearance of labeled 2-deoxy-D-glucose in the intracellular pools, have confirmed previous reports that 2-deoxy-D-glucose first appears in the sugar phosphate pool . Such results are consistent with a transport associated phosphorylation mechanism . Since wild-type yeasts contain three enzymes which could participate in such a process, hexokinase isozymes PI and PII and glucokinase, pool labeling experiments were carried out with single-kinase mutant strains containing only one of these enzymes . Results similar to those for wild-type strains were obtained for all three single-kinase strains, suggesting that if transport associated phosphorylation does occur in baker's yeast, it is not a function of the specific kinase present in the cell . While the results of the pool labeling experiments are consistent with a transport associated phosphorylation mechanism for 2-deoxy-D-glucose, caution is urged in interpreting the results of experiments with whole cells where problems of compartmentation and multiple pools are difficult to assess.

Biokhimiia, 1982 Jun, 47(6), 993 - 8
{Isolation and catalytic properties of the soluble monomeric form of inorganic pyrophosphatase from baker's yeast}; Kasho VN et al.; Data from sedimentation analysis suggest that modification of about 40% of free amino groups of inorganic pyrophosphatase by maleic anhydride, pH 10.5, results in a loss of the enzyme ability to form dimers at neutral values of pH . The specific activity of monomeric pyrophosphatase is 50-80% of that of the dimeric form . The monomer has a pH optimum of about 7, requires metal ions for activation of both enzyme and substrate and is capable of exergonic synthesis of PPi in the active center . The enzyme binding to PPi is strongly stabilized by fluoride . The experimental data indicate that the individual subunit of inorganic pyrophosphatase possesses all the main catalytic properties of native dimeric molecule.

Arch Dis Child, 1982 May, 57(5), 343 - 6
Defective yeast opsonisation and functional deficiency of complement in sickle cell disease; Larcher VF et al.; Opsonisation of heat-killed baker's yeast, functional activity of the total alternative pathway of complement, and factor B detected functionally and immunochemically were significantly reduced in 72 children with sickle cell disease compared with 40 age-matched black control children . There was significant correlation between functional activity of the total alternative pathway and functionally measured factor B, but not between factor B measured functionally and immunochemically . The opsonisation defect could be corrected in vitro by normal serum, and factor B-depleted serum, and was qualitatively similar to that seen in patients with primary yeast opsonisation deficiency . Serial studies showed that these serum defects were persistent . Reduction in the activity of components of the alternative pathway of complement and opsonisation was found in 4 patients who had recovered from pneumococcal meningitis and in one who developed osteomyelitis . Defects of yeast opsonisation and complement which are common in patients with sickle cell disease, may partly explain the children's increased susceptibility to infection, and might help to identify individuals especially at risk.

Biochem J, 1982 May 1, 203(2), 461 - 70
A stereochemical investigation of the hydrolysis of cyclic AMP and the (Sp)-and (Rp)-diastereoisomers of adenosine cyclic 3':5'-phosphorothioate by bovine heart and baker's-yeast cyclic AMP phosphodiesterases; Jarvest RL et al.; Bovine heart cyclic AMP phosphodiesterase, which has a requirement for Mg2+, hydrolyses cyclic AMP with inversion of configuration at the phosphorus atom, but only the (Sp)-diastereoisomer of adenosine cyclic 3':5'-phosphorothioate is hydrolysed by this enzyme . By contrast, the low-affinity yeast cyclic AMP phosphodiesterase, which contains tightly bound Zn2+, hydrolyses both the (Sp)- and the (Rp)-diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, the (Rp)-diastereoisomer being the preferred substrate under V max . conditions . Both of the diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, as well as cyclic AMP, are hydrolysed with inversion of configuration at the phosphorus atom by the yeast enzyme . It is proposed that, with both enzymes, the bivalent metal ion co-ordinates with the phosphate residue of the substrate, and that hydrolysis is catalysed by a direct "in-line' mechanism.

Z Naturforsch {C}, 1982 May-Jun, 37(5-6), 445 - 7
Anomalous reduction of cytochrome b in highly purified complex III from baker's yeast; de la Rosa FF et al.; In highly purified bc1-complex from baker's yeast, the reduction of cyt c1 and partial reduction of cyt b is obtained by catalytic amount of succinate dehydrogenase and succinate in the presence of 7 microM antimycin . After the addition of ferricyanide the c1 is re-oxidized and a increase in the reduction of b is observed . Using stopped-flow we established that the oxidation of c1 by ferricyanide proceeds as a pseudo-first order reaction and the reduction of b is faster and with two phases . Our observation suggests that these two processes are not directly interconnected and that other component than c1 must be the "control factor" in the anomalous reduction of cyt b . This component must be, by exclusion, the iron-sulfur protein.

Hoppe Seylers Z Physiol Chem, 1982 Apr, 363(4), 365 - 73
Arginyl-tRNA synthetase from Escherichia coli K12: specificity with regard to ATP analogs and their magnesium complexes; Gerlo E et al.; Fifteen analogs of ATP have been tested in the ATP/PPi pyrophosphate exchange and the aminoacylation of arginyl-tRNA synthetase from E . coli K12 . Six compounds are substrates in both reactions, whereas seven of the triphosphates were inhibitors for both reactions . The Km, V and Ki values have been determined . The enzyme is less specific against base modifications of the ATP molecule than arginyl tRNA synthetase from baker's yeast in the aminoacylation and is inhibited by more base-modified compounds in the ATP/PPi exchange . The enzyme accepts 3'-deoxy-ATP as substrate and is inhibited by 2'-methoxy-ATP, whereas the reversed observation is made for the yeast arginyl-tRNA synthetase . The stoichiometry and association constants of complexes formed by six ATP analogs (four substrates and two inhibitors) and magnesium ions were investigated; five analogs form 1:1 complexes, one analog (3'-deoxy-ATP) binds two magnesium ions . The enzyme must accept different complexes formed with one or two magnesium ions as substrate, which must be different in structure from complexes proposed in literature.

Eur J Biochem, 1982 Mar 1, 122(3), 553 - 8
Bromopyruvate as an affinity label for Baker's yeast flavocytochrome b2 . Identification of an active-site cysteine and characterization of some cysteine peptides; Alliel PM et al.; It was previously reported that bromopyruvate behaves as an active-site-directed reagent for flavocytochrome b2 {Mulet and Lederer (1977) Eur . J . Biochem . 73, 443-447}, but that some unspecific labeling also took place {Alliel, Mulet, and Lederer (1980) Eur . J . Biochem . 105, 343-351} . In this work, radioactive peptides were purified after labeling the enzyme with bromo{2-14C}pyruvate . Direct proteolysis of the labeled enzyme led to a multiplicity of labeled peptides, due to incomplete proteolysis . Four of them were characterized, corresponding to two unique cysteine residues . Cyanogen bromide cleavage of the labeled protein, followed by enzymatic digestion, led to the isolation of peptides corresponding to four cysteines, including the two previously identified ones . Comparison of the specific radioactivity of the various labeled peptides lead us to the conclusion that the active-site cysteine must be the one present in the 85-residue cyanogen bromide peptide alpha CB3 . The sequence around that cysteine is Ala-Ser-Cys-Ser-Pro-Gln-Gln-Ile-Ile-Glu-Ala-Ala-.

Biokhimiia, 1982 Mar, 47(3), 361 - 73
{Effect of specific antibodies on association of glyceraldehyde 3-phosphate dehydrogenase subunits}; Cherednikova TV et al.; The rabbit antibodies against glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from baker's yeast or rat muscle are strictly specific to the corresponding antigens and are not involved in the cross reaction . The interaction of specific antibodies with Fab-fragments does not affect the activity of the yeast enzyme which is in agreement with the previous data on the rat muscle enzyme . The antibodies against yeast dehydrogenase immobilized on BrCN-activated Sepharose were used for the preparation of the enzyme linked to the matrix by the antigen--antibody complex . The tetrameric enzyme molecule thus immobilized completely retains its activity and the ability to dissociate into dimers in the cold in the presence of ATP . The dimer which remains bound within the antigen--antibody complex retains its activity in the presence of agents causing inactivation and release of the second dimer into solution . The tetramer of yeast dehydrogenase covalently bound to the BrCN-activated Sepharose by one subunit is completely stable under conditions causing dissociation, when it exists in the complex with three molecules of specific antibodies . The binding of four molecules of Fab-fragments of specific antibodies per molecule of tetramer protects the rat muscle apoenzyme against anion-dependent cold inactivation and thermal inactivation in solution . The binding of two antibody molecules specific to rat muscle glyceraldehyde 3-phosphate dehydrogenase to the hybrid tetramer made up of yeast dehydrogenase dimer covalently linked to Sepharose and of rat muscle dehydrogenase dimer, prevents the dissociation of the tetramer (i.e . release of the "muscle" type dimer into solution) . Gel filtration through Sepharose 6B of the apo-glyceraldehyde phosphate dehydrogenase complex with the Fab-fragments of specific antibodies demonstrated that the binding of the Fab-fragments shifts the tetramer in equilibrium or formed from dimers equilibrium in the apoenzyme solution towards the tetramer . It is concluded that the specific antibodies increase the intersubunit interactions in the oligomer by stabilizing the native tertiary structure of individual subunits.

J Biol Chem, 1982 Feb 25, 257(4), 1565 - 8
Histone acetylation in baker's yeast . Maintenance of the hyperacetylated configuration in log phase protoplasts; Nelson DA; The extent and rates of histone acetylation in the yeast Saccharomyces cerevisiae were investigated . The yeast histones are highly acetylated during log phase . The modification examined is not a function of polypeptide synthesis, but rather occurs on the histones subsequent to deposition . The log phase yeast histones are neither rapidly acetylated nor rapidly deacetylated . Acetylation and deacetylation rates for histones H3 and H4 are on the order of hours, indicating that, during log phase, these histones are maintained in the hyperacetylated form . The acetate turnover rate for H2B is somewhat increased over that observed for H3 and H4.

Z Allg Mikrobiol, 1982, 22(7), 443 - 51
{Arthrobacter globiformis - a new yeast-lysing bacterium}; Blechschmidt D et al.; A bacterium capable to lyse viable yeast cells was isolated from compost enriched with baker's yeast cells and was identified as Arthrobacter globiformis . The yeast lytic enzyme complex produced by shaking culture was precipitated with ammonium sulfate, dialyzed and lyophilized . The crude residue contained beta-glucanase and alpha-mannanase, yet not chitinase . Optimale carbon sources in the culture medium for a high enzyme synthesis were 0.5% beta-glucan for the production of beta-glucanase, resp., 3% alpha-mannan for the production of alpha-mannanase . Addition of 10% whole died baker's yeast cells to the culture medium effected similar results . The crude enzyme residue released among other things spheroplasts from cells of the yeast Pichia membranaefaciens, Metschnikowia pulcherrima, Hansenula anomala and Candida guilliermondii "H" . However, it did not or only weakly lyse viable cells of the yeasts Rhodotorula rubra, Rhodosporidium toruloides and Sporobolomyces roseus . The mutants of Candida guilliermondii "H" with modified glucan, resp., mannan concentrations in the cell walls did not indicate differences in their susceptibility to lysis.

Microbios, 1982, 33(132), 111 - 8
An integrative approach to the electrophoresis of the reductase and dehydrogenase isozymes in respiring and fermenting yeasts; Irwin D et al.; A method is described for qualitatively monitoring changes in the starch gel electrophoresis isozyme patterns of key yeast dehydrogenase and reductase activities involved in the transition from aerobic to anaerobic conditions during batch fermentations . The hypoxic transition was accompanied by the disappearance of catalase, superoxide dismutase, mitochondrial dehydrogenases and the appearance of a novel 6-phosphogluconic acid dehydrogenase activity . Significant genetic differences were noted in the banding patterns of wine yeasts, baker's yeast and a commonly used laboratory yeast strain.

Agents Actions Suppl, 1982, 10, 185 - 94
Renal excretion of vanillylmandelic acid and homovanillic acid in rats with paw edema or adjuvant arthritis; Krause E et al.; 1 . Urinary excretion of unconjugated vanillylmandelic acid (VMA) and homovanillic acid (HVA) was found to be decreased in adjuvant arthritic rats . The decrease is apparently not due to impaired animal activity, but it could be explained by the reduction of catecholamine biosynthesis and/or release induced by E prostaglandins the biosynthesis of which is increased in the inflammation . It could also be caused by an increased "consumption" of catecholamines during prostaglandin biosynthesis . 2 . Both the reduction of biosynthesis or release and an increased "consumption" of catecholamines would mean that inflammation is characterized by deficiency of the anti-inflammatory catecholamines . 3 . The investigations failed to demonstrate an adequate decrease of the urinary excretion of VMA and HVA in rats with edemas induced by carrageenin, serotonin, formaline, silver nitrate, kaolin, dextran and baker's yeast, respectively . The experiments should be repeated with other catecholamine metabolites.

What Is Bioassay?, What Is Biotechnology?, What Is Genome?, What Is Yeast?, What Is Bioremediation?, e, Bacteriology, o, Microbe, s, Microorganisms, r, Microorganism, i, Microbiology, i, Microorganisms, a, Meningococcus, o, Salmonella typhimurium, n, Antibiotics, s, Escherichia coli, n, Escherichia coli, e, Clostridia, r, Multidrug resistant, a, Meningococcus, n, S. cerevisiae, s, Clostridia, n, S. cerevisiae, c, Cryptococci, c, Meningococcus, c, Pseudomonas aeruginosa, s, Cryptococci, r, Halomonas, s, Bactericidal, i, Multidrug resistant, o, E coli O157, s, Biocontactor

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005