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