Yeast

Acta Biol Med Ger, 1982, 41(11), 991 - 1002
A new procedure for the purification of proteinase B from baker's yeast and interaction of the purified enzyme with a specific inhibitor; Huse K et al.; Proteinase B from baker's yeast was purified to homogeneity by applying affinity chromatography with D,L-tyrosine ethyl ester as ligand . The molecular parameters of the product are similar to those reported formerly by other groups . A different form of proteinase B is isolated if affinity chromatography is replaced by CM-cellulose chromatography and gel filtration . In this case a peptide tightly associated with the enzyme was found to occur . This could be identified as an inhibitor fragment produced by limited proteolysis of a proteinase B bound inhibitor induced by proteinase A.

Folia Microbiol (Praha), 1982, 27(4), 217 - 21
Maltotriose transport and utilization in baker's and brewer's yeast; Michaljanicova D et al.; Maltotriose is metabolized by baker's and brewer's yeast only oxidatively, with a respiratory quotient of 1.0, the QCO2Ar being, depending on the strain used, 0-11, as compared with QCO2air of 6-42 microL CO2 per h per mg dry substance . The transport appeared to proceed by facilitated diffusion (no effects of NaF, iodoacetamide and 3-chlorophenylhydrazonomalononitrile) with a KT of more than 50 mM and was inhibited by maltose greater than maltotriose greater than methyl-alpha-D-glucoside greater than maltotetraose greater than D-fructose greater than D-glucose . The transport was present constitutively in both Saccharomyces cerevisiae (baker's yeast) and in S . uvarum (brewer's yeast) and it was not significantly stimulated by preincubation with glucose or maltose . The pH optimum was 4.5-5.5, the temperature dependence yielded an activation energy of 26 kJ/mol.

Infect Immun, 1981 Dec, 34(3), 980 - 6
Participation of pili and cell wall adhesion in the yeast agglutination activity of Escherichia coli; Eshdat Y et al.; Escherichia coli strain 2699 (O6:K13) which had been isolated from a case of urinary tract infection exhibited pili during the stationary phase (24 to 40 h), but not during the exponential phase (4 h), when grown in static broth culture . The bacteria were also piliated when grown for 24 h on agar . They agglutinated Saccharomyces cerevisiae (baker's yeast) in the piliated as well as in the nonpiliated state . The agglutinations were mannose sensitive, i.e., they could be inhibited with 50 mM methyl-alpha-mannoside . The bacteria were first depiliated by shearing and then used for the isolation of outer membrane vesicles with an Omnimixer . Purified pili and outer membranes were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy . The pili could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis only after treatment at low pH or in saturated guanidine hydrochloride which is typical of the common type 1 pili . Depiliated bacteria, purified pili, and purified outer membranes gave mannose-sensitive agglutination of S . cerevisiae . The findings are discussed with respect to possible mechanisms of cell agglutination.

Biochim Biophys Acta, 1981 Nov 13, 662(1), 165 - 7
Purification and properties of guanylate kinase from baker's yeast; Moriguchi M et al.; Guanylate kinase (ATP:(d)GMP phosphotransferase, EC 2.7.4.8) was purified about 200-fold with 4% yield from baker's yeast . The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was calculated to be 25 000 by gel filtration . With ATP as a phosphate donor, the kinase used only GMP as a phosphate acceptor . Km values for ATP and GMP were 0.5 and 0.048 mM, respectively . The enzyme reacted optimally at pH 7.5 . The enzyme was labile during storage at 4 degrees C and inactivation was prevented by 20% glycerol.

Clin Exp Immunol, 1981 Nov, 46(2), 412 - 9
A study of C3b deposition on yeast surfaces by sera of known opsonic potential; Turner MW et al.; C3c fragments released by the actin of trypsin from C3b molecules bound to zymosan may be readily quantitated by conventional gel techniques to give a measurement of the efficiency of C3b opsonization . Using this approach, the rate of deposition of C3b molecules was found to be very rapid in sera known to opsonize yeast normally . In contrast, sera defective in yeast opsonization deposited C3b much more slowly . The C3b elution technique was found to correlate well with both a direct phagocytosis assay using baker's yeast (r = 0.87, P less than 0.001) and a recently described neutrophil iodide uptake assay (r = 0.88, P less than 0.001).

Clin Exp Immunol, 1981 Nov, 46(2), 406 - 11
Mechanism of the serum defect in yeast opsonization in children with fulminant hepatic failure (FHF); Larcher VF et al.; Defective opsonization of heat-killed baker's yeast was found in all 14 children with fulminant hepatic failure (FHF) but returned to normal in two who recovered and was normal in six mothers of patients . Yeast opsonization was significantly correlated with factor B activity of FHF serum but not with other components of classical or alternative pathways of complement . Reconstitution and family studies suggested that defective yeast opsonization in FHF is secondary and, although dependent on factor B activity, is qualitatively and quantitatively similar to that of primary yeast opsonization deficiency . These findings suggest that the factors responsible for opsonization of yeasts, or their regulation, are synthesized or controlled by the liver.

Eur J Biochem, 1981 Nov, 120(2), 279 - 87
Study of a zone highly sensitive to proteases in flavocytochrome b2 from Saccharomyces cerevisiae; Ghrir R et al.; Flavocytochrome b2 from baker's yeast is a bifunctional tetrameric protein which carries two prosthetic groups, FMN and heme, per subunit of Mr 58 000 . The amino terminus of the subunit is wrapped around the heme and constitutes the so-called cytochrome b2 core (Mr 11 000), homologous to cytochrome b5 . It has been shown in the past that a number of proteases (yeast proteases, chymotrypsin) preferentially cleave the peptide chain at a point situated much further down the polypeptide chain than the C terminus of the heme-binding domain . Some enzymatic parameters are concomitantly modified, but not the quaternary structure . This paper describes the conditions for selective proteolysis of intact flavocytochrome b2 and of its various previously studied stable nicked forms by the protease from Staphylococcus aureus V8 . Successive attack by a combination of two proteases is also described . We have established the amino acid sequence of the area where proteolytic attack takes places, and shown that chymotrypsin and S . aureus protease open only one bond, whereas yeast proteases remove five residues from the central part . The various nicked forms, some of which have lost up to 16 amino acid residues, have been enzymatically characterized . These and previous results lend support to, but do not prove, the idea that the flavodehydrogenase part of flavocytochrome b2 may be composed of two domains, linked by the region accessible to proteases . That area might constitute a hinge or rather a clasp between the domains.

Eur J Biochem, 1981 Oct, 119(3), 581 - 7
Pyruvate dehydrogenase complex from baker's yeast . 2 . Molecular structure, dissociation, and implications for the origin of mitochondria; Kresze GB et al.; 1 . Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (s20,w 77 S) and flavin content (1.3--1.4 nmol/mg) to the complexes from mammalian mitochondria . 2 . The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58 000; lipoamide dehydrogenase (E3), 56 000; pyruvate dehydrogenase (E1), alpha-subunit, 45 000, and beta-subunit, 35 000 . Gel chromatography in the presence of 6 M guanidine . HCl gave a value of 52 000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes . Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria . 3 . The complex was resolved into its component enzymes by the following methods . E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution . E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea . The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.

Eur J Biochem, 1981 Oct, 119(3), 573 - 9
Pyruvate dehydrogenase complex from baker's yeast . 1 . Purification and some kinetic and regulatory properties; Kresze GB et al.; Pyruvate dehydrogenase complex, for the first time, was highly purified from commercial baker's yeast (saccharomyces cerevisiae) . Proteolytic degradation was prevented by the inclusion of the protease inhibitors pepstatin A, leupeptin, and phenylmethanesulfonyl fluoride during the enzyme purification . The yield from 1 kg of pressed yeast was about 15--20 mg enzyme with a specific activity of 17--30 U/mg . Most of the kinetic and regulatory properties of the yeast enzyme were found similar to those of the mammalian mitochondrial pyruvate dehydrogenase complexes except that Km for pyruvate, when assayed at the pH optimum, was much higher than in the mammalian complexes and resembled the values reported for the complexes of gram-negative bacteria . Furthermore, neither in yeast homogenates nor in the isolated yeast pyruvate dehydrogenase complex, was any evidence found for regulation by interconversion (phosphorylation-dephosphorylation) as occurs in mammals, plants, and Neurospora crassa.

Eur J Biochem, 1981 Oct, 119(2), 395 - 400
Large-scale purification and phosphorylation of a detergent-treated adenosine triphosphatase complex from plasma membrane of Saccharomyces cerevisiae; Foury F et al.; A new procedure for large-scale preparation of plasma-membrane-bound ATPase from Saccharomyces cerevisiae is described . The crude membrane fraction is purified by selective extraction with three successive detergents: deoxycholate (0.25 mg/mg protein), Triton X-100 (0.25%) and lysophosphatidylcholine (1 mg/mg protein) . These treatments extract the mitochondria and strip the plasma membrane . From 1 kg commercial baker's yeast, 200 mg of plasma membrane proteins are isolated in 2--3 days . Plasma-membrane-bound ATPase of specific activity of 10--13 mumol Pi x min-1 x mg protein-1 is obtained with a yield estimated to 60% . Dodecylsulfate/polyacrylamide gel electrophoresis shows three predominant polypeptides of Mr = 95000, 70000 and 56000 in the purified membrane fraction . The major polypeptide of Mr = 95000 identified as the ATPase subunit is phosphorylated by millimolar concentrations of ATP . The phosphorylated intermediate reaches the steady-state level in less than 100 ms and turns over very rapidly . It is hydrolyzed by hydroxylamine . Its formation is prevented by the ATPase inhibitors vanadate and Dio-9, a plasma-membrane ATPase inhibitor of unknown structure . At least four other membrane proteins are phosphorylated with much slower kinetics, presumably through the action of protein-kinase(s).

Eur J Biochem, 1981 Oct, 119(3), 477 - 82
Arginyl-tRNA synthetase from Baker's yeast . Order of substrate addition and action of ATP analogs in the aminoacylation reaction; influence of pyrophosphate on the catalytic mechanism; Freist W et al.; The order of substrate addition to arginyl-tRNA synthetase from baker's yeast has been investigated by bisubstrate kinetics, product inhibition and inhibition by three different inhibiting ATP analogs, the 6-N-benzyl, 8-bromo and 3'-deoxy derivatives of ATP, each acting competitively with respect to one of the substrates . The kinetic patterns are consistent with a random ter-ter mechanism, an addition of the three substrates and release of the products in random order . The different inhibitors are bound to different enzyme . substrate complexes of the reaction sequence . Addition of inorganic pyrophosphatase changes the inhibition patterns and addition of methylenediphosphonate as pyrophosphate analog abolishes the effect of pyrophosphatase, showing that the concentration of pyrophosphate is determinant for the mechanism of catalysis.

Biochim Biophys Acta, 1981 Sep 15, 661(1), 136 - 41
The substrate specificity of proteinase B from baker's yeast; Kominami E et al.; The substrate specificity of proteinase B (EC 3.4.22.9) from Baker's yeast was studied . Experiments with unblocked synthetic peptides indicated that the enzyme has no aminopeptidase activity . The proteinase cleaves trypsin substrates like Bz-Arg-OEt, Bz-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA and chymotrypsin substrates like Ac-Tyr-OEt and Bz-Tyr-pNA . The Km value for Ac-Tyr-OEt is similar to that of chymotrypsin A, but the catalytic activity per mol proteinase B amounts to only 1/20 that of chymotrypsin A . Km and kcat for Bz-Arg-OEt are 1/50 and 1/7 as high as the corresponding values determined for trypsin . Proteinase B cleaved the oxidized insulin B chain with an initial rapid cleavage step at Leu(15)-Tyr(16) and Phe(24)-Phe(25) . Slower hydrolysis was observed at Gln(4)-His(5), Leu(11)-Val(12) Tyr(16)-Leu(17), Leu(17)-Val(18), Arg(22)-Gly(23) and Phe(25)-Tyr(26) . These results suggest that the specificity of proteinase B is comparable to the specificity of porcine chymotrypsin C as well as of trypsin . When the hexapeptide Leu-Trp-Met-Arg-Phe-Ala was used as a substrate for proteinase B, the enzyme preferentially attacked at Arg-Phe and more slowly at Trp-Met.

Biochim Biophys Acta, 1981 Sep 15, 661(1), 124 - 35
Purification and properties of proteinase B from yeast; Kominami E et al.; Proteinase B (EC 3.4.22.9) was purified from commercial baker's yeast and from wild type strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis . For large scale purification a procedure was developed involving hydrophobic chromatography on octyl-Sepharose 4B and gel filtration on Sephadex G-100 . A rapid purification of small amounts of proteinase B was achieved by affinity chromatography on the nitrated proteinase B inhibitor, immobilized on CH-Sepharose according to Bunning and Holzer (Bunning, P . and Holzer, H . (1977) . J . Biol . Chem . 252, 5316-5323) . The enzyme prepared from all three sources appeared to be homogeneous and exhibited a molecular weight of 33 000 in SDS-polyacrylamide gel electrophoresis . Homogeneity and molecular weight were confirmed for the enzyme from baker's yeast by ultracentrifugation studies . Polyacrylamide gel electrophoresis without SDS and electrofocusing however, indicated microheterogeneity of the proteinase B activity . The aminoterminal residue of the enzyme was found to be glycine . Proteinase B turned out to be a glycoprotein, containing 8-9% neutral sugars and 1.5% amino sugars . The enzyme is blocked by p-hydroxymercuribenzoate and by the serine proteinase inhibitors DFP and PMSF . Among the proteinase inhibitors from microbial origin, chymostatin and antipain were the most powerful inhibitors of proteinase B.

Hoppe Seylers Z Physiol Chem, 1981 Sep, 362(9), 1247 - 54
Survey on substrate specificity with regard to ATP analogs of aminoacyl-tRNA synthetases from E . coli and from Baker's yeast . Correlation to synthetase families; Freist W et al.; The substrate specificity of twenty aminoacyl-tRNA synthetases from E . coli and thirteen enzymes from baker's yeast with regard to eight ATP analogs is investigated for a comparison of the active-site topography . The enzymes are arranged in a scheme of possible "enzyme families" and compared to earlier schemes.

Eur J Biochem, 1981 Sep, 119(1), 151 - 64
Isoleucyl-tRNA synthetase from Baker's yeast . Action of ATP analogs in pyrophosphate exchange and aminoacylation, two pathways of the aminoacylation depending on concentration of pyrophosphate; Freist W et al.; The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates . The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase . Additionally, bisubstrate kinetics and product inhibition studies were carried out . The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates . The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine . For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong . Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.

Biokhimiia, 1981 Sep, 46(9), 1674 - 80
{Stable compound of inorganic pyrophosphatase with pyrophosphate obtained by a fluoride-mediated reaction with phosphate}; Bakuleva NP et al.; Incubation of inorganic pyrophosphate from baker's yeast with phosphate and MgCl2 in the presence of fluoride results in a gradual inactivation of the enzyme concomitant with incorporation of PP1 (about 2 moles per mole) into the protein . The rate constant for this process shows an increase with a rise in concentrations of the three reagents, the maximal value of inactivation being 0.11 min-1 . The bound PP1 is not separated by gel-filtration . The rate of spontaneous degradation of the enzyme-pyrophosphate complex and the nature of EDTA and Mg2+ effects are similar to those for the analogous compound obtained by inhibition of PP1 hydrolysis by fluoride . The data obtained suggest that during PP1 synthesis and hydrolysis by pyrophosphatase fluoride stabilizes the same intermediate of the enzyme with pyrophosphate.

Biochemistry, 1981 Aug 4, 20(16), 4693 - 7
Thermodynamics of the binding of D-glucose to yeast hexokinase; Takahashi K et al.; The binding of D-glucose to baker's yeast hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) was studied by isothermal and differential scanning calorimetry (DSC) and by fluorometric titration . The enthalpy and heat capacity changes associated with the binding of glucose were found to be nearly zero at both low and high ionic strengths over the temperature range from 7 to 29 degrees C . Thus, the free-energy change, amounting to -5.1 kcal mol(-1) at 25 degrees C and high ionic strength, is nearly independent of the temperature and is primarily of entropic origin . DSC study of the thermal unfolding of the free enzyme at low ionic strength gave an excess heat capacity curve with two maxima . This result appears to reflect a difference in thermal stability of the two domains in the hexokinase molecule which are indicated by X-ray crystallography {Bennett, W.S., & Steitz, T . A . (1978) Proc . Natl . Acad . Sci . U.S.A . 75, 4848-4852} . In contrast, the unfolding of free enzyme at high ionic strength was fully cooperative . The excess heat capacity curve for the unfolding of the glucose-bound enzyme had only one peak at both low and high ionic strengths . This is consistent with the X-ray result that the binding of glucose induces a conformational change in the enzyme which brings the two lobes into close proximity . It is interesting that such a significant, molecule-wide conformational change is accompanied by only very small net changes in enthalpy and heat capacity.

J Bioenerg Biomembr, 1981 Aug, 13(3-4), 171 - 9
The partially reduced species present in purified cytochrome oxidase from baker's yeast is cytochrome a; Siedow JN et al.; Cytochrome oxidase purified from baker's yeast submitochondrial particles is found to exist in a partially reduced state in the resting enzyme . Studies utilizing optical and EPR spectroscopy indicate that the "inactive" fraction contains a reduced low-spin heme, cytochrome a possibly indicating a block of electron transfer from cytochrome a to cytochrome a 3 . There is no apparent reduction of either the EPR-detectable copper or the species associated with the 830 nm band . Oxidative titrations of the resting-state yeast cytochrome oxidase indicate that the reduction potential of the species titrating is higher than that of ferricyanide . This "inactive" cytochrome oxidase is not the result of the isolation procedure, but seems to represent a species which is present in the intact yeast.

Am J Trop Med Hyg, 1981 Jul, 30(4), 762 - 8
Immunization against Leishmania donovani: glucan as an adjuvant with killed promastigotes; Holbrook TW et al.; Mice were immunized by a series of intravenous injections of formalin-killed Leishmania donovani promastigotes alone and combined with glucan, a beta 1,3 polyglucose derivative of baker's yeast . In three separate experiments animals were challenged with viable parasites on day 21, 40 or 80 after immunization . Mice which received dead parasites and glucan exhibited resistance against challenge up to 80 days after immunization . Animals which had been injected with glucan alone exhibited a lesser degree of resistance but injections of killed promastigotes alone conferred no measurable resistance against infection.

Biochemistry, 1981 Jun 23, 20(13), 3851 - 6
Phenylalanyl-tRNA synthetase of baker's yeast . Modulation of adenosine triphosphate-pyrophosphate exchange by transfer ribonucleic acid; Fasiolo F et al.; Native and modified phenylalanine transfer ribonucleic acid (tRNAPhe) can modulate phenylalanine-dependent adenosine triphosphate--inorganic {32P}pyrophosphate (ATP--{32P}PPi) exchange activity via inhibition of adenylate synthesis . Inhibition is visualized if concentrations of L-phenylalanine, ATP, and pyrophosphate are subsaturating . In the proposed mechanism, tRNAPhe is a noncompetitive inhibitor at conditions where only one of the two active sites per molecule of enzyme is occupied by L-phenylalanine, ATP, and pyrophosphate . At saturating concentrations of these reactants, both active sites are occupied and, according to the model, inhibition is eliminated . Occupation by these reactants is assumed to follow homotropic negative cooperativity . The type of effects depends on modification of tRNAPhe . Native tRNAPhe, tRNA2'-dAPhe, and tRNAoxi-redPhe are inhibitors, tRNAPhepCpC has no effect, and tRNAoxPhe is an activator . Kinetics of activation by tRNAoxPhe are slow, following the time course of Schiff base formation and subsequent reduction by added cyanoborohydride . Besides showing that a putative enzyme amino group is nonessential for substrate binding and adenylate synthesis, this result may suggest that an enzyme amino group could interact with the 3'-terminal adenyl group of cognate tRNA . In the case of asymmetrical occupation of the enzyme active sites by all of the small reactants ATP, L-phenylalanine, and pyrophosphate, the interaction with the amino group might trigger the observed noncompetitive inhibition of the pyrophosphate exchange by tRNAPhe.

Eur J Cell Biol, 1981 Jun, 24(2), 216 - 25
Isolation and characterization of paracrystalline arrays of the plasma membrane of baker's yeast Saccharomyces cerevisiae; Maurer A et al.; Yeast plasma membranes were isolated from homogenized cells and analyzed by SDS-PAGE . Two glycoproteins of 160 000 and 240 000 molecular weight were found, both of which exhibited invertase activity (EC 3.2.1.26) . By density gradient centrifugation a heavy membrane fraction which consisted of the glycoproteins and two hydrophobic proteins was isolated . Antibody labeling of protoplasts revealed a good correlation between the distribution of binding sites of the antibodies against the heavy fraction and the distribution of the intramembranous particles . The cytoplasmic surface of the yeast plasma membrane was visualized by freeze drying and subsequent platinum/carbon shadowing of membrane vesicles adsorbed to cationized glass and squirted with a hypotonic buffer stream . In contrast to the smooth exoplasmic surface the cytoplasmic surface showed paracrystalline arrays of particles which resembled in size, number and lattice constant the intramembranous particles . Removal of the adsorbed paracrystalline arrays and subsequent SDS-PAGE revealed the same protein pattern as the heavy membrane fraction . It can therefore be concluded that the glycoproteins which show invertase activity and the two hydrophobic proteins are the major components of the paracrystalline arrays . It is proposed that the glucose level of the nutrient medium influences the appearance and disappearance of the paracrystalline arrays, which consist mainly of invertase, because synthesis of invertase is inhibited by glucose levels higher than 1%.

Biochemistry, 1981 Apr 14, 20(8), 2065 - 74
Catalytic mechanism of valyl-tRNA synthetase from baker's yeast . Reaction pathway and rate-determining step in the aminoacylation of tRNAVal; Kern D et al.; The catalytic mechanism of valyl-tRNA synthetase from baker's yeast has been investigated by pre-steady-state and steady-state kinetic measurements and end product dissociation studies . The pre-steady-state kinetics show a lag period during the early time when the reaction is started with free enzyme . The preincubation of the synthetase with tRNAVal and/or valine or preformation of Val approximately AMP leads to a progressive suppression of the lag . This lag probably reflects conformational transitions of the enzyme-substrate complex necessary for the transfer . At low pH or at a low ionic strength, the tRNAVal charging occurs much faster at the pre steady state than at the steady state . We show that after the fast transfer of valine from adenylate to tRNAVal, followed by the fast dissociation of AMP and PPi, a new adenylate is synthesized which promotes the dissociation of the nascent Val-tRNAVal . This dissociation occurs in a multistep process . First ATP and magnesium promote the ejection of the valine moiety of Val-tRNAVal from the adenylate site . A new adenylate is then synthesized which promotes, in the presence of magnesium, several state changes of the end product complex . A complex is finally generated in which the enzyme-bound Val-tRNAVal is able to exchange rapidly with a tRNAVal molecule . The free tRNAVal plays an active role in this exchange . Depending upon the experimental conditions, one of these steps can determine the steady-state rate of tRNAVal charging . The dissociations of enzyme-bound uncharged tRNAVal or aa-tRNAs substituted on the amino acid or on the tRNA parts by noncognate parts as well as the effect of the replacement of the adenylates by wrong adenylates have been investigated . It is shown that the valine and the tRNA moieties of Val-tRNAVal and the valine moiety of the adenylate are involved in this mechanism of dissociation . Finally, the rate-determining step of the reversal of tRNAVal charging at the steady-state has been investigated . It is shown that this step is the dissociation of the deacylated tRNAVal from enzyme.

Biochim Biophys Acta, 1981 Mar 26, 653(1), 83 - 90
Purification and some properties of alanyl- and leucyl-tRNA synthetases from baker's yeast; Kern D et al.; Alanyl- and leucyl-tRNA synthetases from baker's yeast were purified to homogeneity in the presence of the protease inhibitor phenylmethylsulfonyl fluoride . Both consist of single polypeptide chains of 118 000 and 125 000 daltons, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions . The monomeric structure of leucyl-tRNA synthetase differs from the dimeric one obtained previously in the absence of protease inhibitors . This illustrates the sensitivity of the synthetases to proteolytic actions and indicates that native structures can only be obtained under optimal protecting conditions . Alanyl- and leucyl-tRNA synthetases differ with respect to pH optimum (6.5 and 8.5, respectively), Michaelis constant for amino acid (1 mM and 0.03, respectively) and in the rate-limiting step for the tRNA aminoacylation reaction . Whereas the catalytic step itself was rate-limiting for alanyl-tRNA synthetase, a step occurring after this was rate-limiting for leucyl-tRNA synthetase.

Biochim Biophys Acta, 1981 Mar 13, 658(1), 45 - 53
Purification of an acidic alpha-D-mannosidase from Aspergillus saitoi and specific cleavage of 1,2-alpha-D-mannosidic linkage in yeast mannan; Ichishima E et al.; An acidic alpha-D-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) has been isolated from culture filtrate of Aspergillus saitoi . The extracellular alpha-mannosidase was homogeneous in polyacrylamide gel electrophoresis . The molecular weight of the enzyme was 51 000 and the isoelectric point pH 4.5 . The purified enzyme has a pH optimum of 5.0, a Km of 0.45 mM with baker's yeast mannan and has no activity towards p-nitrophenyl-alpha-D-mannoside . The mode of action of the enzyme has been studied with baker's yeast mannan and sake yeast mannan . The enzyme cleaves specifically the 1,2-alpha-linked side chain, producing free mannose.

Biochemistry, 1981 Feb 3, 20(3), 468 - 72
Role of arginine residue in saccharopine dehydrogenase (L-lysine forming) from baker's yeast; Fujioka M et al.; The baker's yeast saccharopine dehydrogenase (EC 1.5.1.7) was inactivated by 2,3-butanedione following pseudo-first-order reaction kinetics . The pseudo-first-order rate constant for inactivation was linearly related to the butanedione concentration, and a value of 7.5 M-1 min-1 was obtained for the second-order rate constant at pH 8.0 and 25 degrees C . Amino acid analysis of the inactivated enzyme revealed that arginine was the only amino acid residue affected . Although as many as eight arginine residues were lost on prolonged incubation with butanedione, only one residue appears to be essential for activity . The modification resulted in the change in Vmax, but not in Km, values for substrates . The inactivation by butanedione was substantially protected by L-leucine, a competitive analogue of substrate lysine, in the presence of reduced nicotinamide adenine dinucleotide (NADH) and alpha-ketoglutarate . Since leucine binds only to the enzyme-NADH-alpha-ketoglutarate complex, the result suggests that an arginine residue located near the binding site for the amino acid substrate is modified . Titration with leucine showed that the reaction of butanedione also took place with the enzyme-NADH-alpha-ketoglutarate-leucine complex more slowly than with the free enzyme . The binding study indicated that the inactivated enzyme still retained the capacity to bind leucine, although the affinity appeared to be somewhat decreased . From these results it is concluded that an arginine residue essential for activity is involved in the catalytic reaction rather than in the binding of the coenzyme and substrates.

Biokhimiia, 1981 Feb, 46(2), 195 - 201
{Quaternary structure of transketolase from rat liver}; Minin AA et al.; The quaternary structure of immobilized rat liver transketolase was studied . It was shown that the individual subunits of the enzyme are active in their specific activity do not differ from the dimeric form of the enzyme . The quaternary structure stabilizes the coenzyme binding to the protein; thiamine pyrophosphate is split off from the choloenzyme only upon destruction of the quaternary structure . The hybrid dimers formed by the subunits of transketolase from rat liver and baker's yeast were obtained . Their formation requires the presence of the coenzyme and Ca2+ in the medium . No reassociation of the rat liver transketolase subunits into a dimer was shown . It was assumed that in vivo the subunits of rat liver transketolase do not exist in a free state and their association into dimers occurs immediately after completion of the polypeptide chain biosynthesis in the presence of thiamine pyrophosphate.

Eur J Biochem, 1981 Feb, 114(2), 255 - 62
Large-scale purification and some properties of malate synthase from baker's yeast; Durchschlag H et al.; 1 . Malate synthase from baker's yeast (5 kg) was purified 400--500-fold to homogeneity . About 50--200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell-free crude extracts . The enzyme, pI = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies . 2 . Sedimentation and diffusion coefficients were determined: S 0 20,w = 8.26 +/- 0.05 S, D 0 20,w = 4.5 +/- 0.1 X 10(-7) cm2 s-1 . The molecular weight of the synthase was found to be 175 000 +/- 10 000 and 180 000 +/- 10 000 by sedimentation/diffusion and by high-speed sedimentation equilibrium respectively . It was concluded from these and other results that malate synthase has a molecular mass of 180 000 +/- 10 000 . 3 . The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66 000 . The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass . 4 . The binding of acetyl-coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+ . Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism . 5 . The intermolecular isotopic effect, kH:k2H = 1.4, was determined with acetyl-coenzyme A and {2H3}acetyl-coenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates . An enzymic procedure for the preparation of doubly labelled {3H, 14C}acetyl-coenzyme A is also presented.

Biochemistry, 1981 Jan 6, 20(1), 122 - 31
Glycyl-tRNA synthetase from baker's yeast . Interconversion between active and inactive forms of the enzyme; Kern D et al.; Glycyl-tRNA synthetase from baker's yeast has been purified to homogeneity . This synthetase was found to be very sensitive to proteases present in the yeast extracts and to oxidizing agents of thiol groups . In the absence of protease inhibitors and/or dithioerythritol, the enzyme rapidly lost its activity and could not be isolated . The use of these protectors allowed us to obtain different oligomeric structures of the synthetase . In the presence of a minimal concentration of dithioerythritol but in the absence of protease inhibitors, a tetrameric glycyl-tRNA synthetase of the alpha 2 beta 2 type (alpha = 67 600, beta = 57 500) with a very low specific activity was recovered . With high concentrations of both protectors, a dimeric enzyme was isolated with a specific activity comparable to that for other yeast synthetases . The enzyme was of the alpha 2 type where alpha = 70 000--80 000 daltons, depending on whether phenylmethanesulfonyl fluoride or diisopropyl fluorophosphate was used as the protecting agent . The native form of the enzyme (alpha 2 = 160 000) associated easily with other proteins in various complexes of molecular weights from 250 000 to 300 000, some of them containing valyl-tRNA synthetase . The dimeric glycyl-tRNA synthetase was found in equilibrium with its subunits . Diluting the enzyme solution or increasing the salt concentration displaced the equilibrium toward the monomers, which are catalytically inactive for both the tRNA aminoacylation and the PPi-ATP exchange reactions . Addition of both tRNAGly and ATP.MgCl2 plus glycine displaced the equilibrium toward the dimeric form of the enzyme . Thiol groups were found to be involved in the association between the two subunits and in both activities of the synthetase . The results are interpreted in the light of possible regulatory mechanisms of the activity of this synthetase.

Int Arch Allergy Appl Immunol, 1981, 64(1), 25 - 41
Mediators of immune-complex-induced aggregation of polymorphonuclear neutrophils . II . Platelet-activating factor as the effector substance of immune-induced aggregation; Camussi G et al.; Platelet-activating factor (PAF) is released in vitro during human and rabbit polymorphonuclear neutrophil (PMN) aggregation induced by C5a anaphylatoxin, neutrophil cationic proteins (CP) and their carboxypeptidase-B-derived fragments, C5a des Arg and CP des Arg, as well as phagocytosis of opsonized baker's yeast particles and immune complexes (IC) . Purified PAF itself is able to cause in vitro PMN aggregation . By using selective inhibitors, we show that PMN aggregation, induced either by PAF or by other soluble stimuli such as C5a, CP and their des Arg products, follows a similar metabolic pathway, which is both adenosine-diphosphate-(ADP)- and arachidonic acid (AA)- independent . The in vivo injection of purified PAF into rabbits leads both to formation of intravascular PMN aggregates and to development of acute neutropenia, which has the same features as those observed after challenge with IC, C5a and CP . In this respect, electron-microscopic studies of intravascular PMN aggregates in the pulmonary capillary network and glomeruli show identical ultrastructural patterns . Moreover, the intravascular release of PAF is demonstrated after the intravenous injection of IC and temporally correlated with the development of neutropenia . We suggest that PAF is probably the final, common, effector substance of IC-, C5a-, C5a-des-Arg-, CP-, CP-des-Arg-mediated PMN aggregation.

Acta Biochim Pol, 1981, 28(3-4), 337 - 49
Effect of sodium ion concentration on transfer RNA conformation in solution studied by Rayleigh light scattering; Patkowski A et al.; The sodium ion concentration dependent conformational changes of transfer RNA (unfractionated tRNA from baker's yeast) have been studied in unbuffered aqueous solutions by Rayleigh light scattering . Changes of the optical parameters of the molecule indicated the following conformational changes of tRNA with increasing NaCl concentration: in salt-free solution tRNA molecules have an irregular hairpin loop-like structure in which the orientation of base rings is not correlated . Upon addition of a small amount of NaCl (0.005 M) an increasing ordering of this structure is observed . In 0.1 M-NaCl the molecule has an extended structure with ordered regions (arms) . Further increase of sodium ion concentration up to 2 M results in folding of the extended structure and formation of a compact and rigid conformation in which most of the bases are nearly perpendicular to the symmetry axis of the molecule.

Folia Microbiol (Praha), 1981, 26(5), 377 - 81
Dependence of phosphate transport in yeast of glycolytic substrates; Knotkova A et al.; Preincubation of baker's yeast (wild strain, respiration-deficient mutant and a low-phosphorus culture) with glucose, trehalose, and other metabolic sugars increases the subsequent uptake of inorganic phosphate 3-5 times . The Kt is reduced by the preincubation from 3.5 to 1.6 mM . The process involves primarily the production of glycolytic energy sources (suppression by iodoacetamide, no effect of antimycin or dicyclohexylcarbodiimide, negligible effect of ethanol, or respiratory mutation) . The low-phosphorus yeast takes up phosphate anions about 1-20 times faster than the high-phosphorus (normal) culture . The stimulation is also accompanied by some (apparently nonessential) protein synthesis and has a halftime of 35 min; its decay has a t0.5 of 12 min but affects only less than one-half of the stimulated capacity.

J Lipid Res, 1981 Jan, 22(1), 171 - 7
Sterol synthesis: a simple method for the isolation of zymosterol (5 alpha-cholesta-8, 24-dien-3 beta-ol) from yeast and spectral properties of zymosterol; Taylor UF et al.; A relatively simple method is described for the isolation of zymosterol (5 alpha-cholesta-8, 24-dien-3 beta-ol) of high purity from baker's yeast . Also presented are detailed spectral properties, including 13C NMR spectral analyses, of zymosterol and its acetate derivative.

Biochim Biophys Acta, 1980 Dec 4, 616(2), 381 - 3
Delta 1-pyrroline-5-carboxylate reductase from baker's yeast: further purification by affinity chromatography with 5' AMP-Sepharose 4B; Matsuzawa T et al.; A homogeneous preparation of delta 1-pyrroline-5-carboxylate reductase (L-proline: NAD (P)+ 5-oxidoreductase, EC 1.5.1.2) was obtained from baker's yeast by an affinity chromatography, using 5' AMP-Sepharose 4B . After the 1st DEAE-Sephadex column chromatography, the enzyme absorbed on 5' AMP-Sepharose column was eluted with 1 mM ATP . The final preparation was homogeneous on polyacrylamide gel electrophoresis and purified 3500-fold from the crude extract . This purified enzyme was very useful as a coupling enzyme for the assays of ornithine and pyrroline-5-carboxylate in tissues, and for the rate assay of ornithine aminotransferase activity.

Eur J Cell Biol, 1980 Dec, 23(1), 6 - 15
Immunochemical analysis of the plasma membrane from baker's yeast Saccharomyces cerevisiae; Baumgartner B et al.; Yeast plasma membranes have been isolated from homogenized yeast cells, identified as pure plasma membrane vesicles which were used as antigens . By crossed immunoelectrophoresis with anti-membrane immunoglobulins, 17 discrete antigens have been detected in Triton X-100 extracts from plasma membranes . Three different immunoabsorption experiments were performed with : a) isolated membranes exposing the cytoplasmic surfaces (PS) and the external surfaces (ES), b) yeast protoplasts exposing only antigenic determinants on the ES, c) lysed protoplasts which had been saturated on the ES with antibodies prior to lysis . These absorption experiments demonstrated that seven of the antigens are expressed on the ES while eight immunogens expose antigenic determinants on the PS . Four of the principal immunoprecipitates are not affected by absorption with surface antigens whereas two of the antigens indicate transmembrane characteristics . Of these 17 immunoprecipitates four were shown by zymograms to possess enzymatic activities: ATPase (EC 3.6.1.3) and NADH-dehydrogenase (EC 1.8.99.3) (three separate components) . Three of these enzymes are expressed on the PS, and one NADH-dehydrogenase exposes determinants on the ES of the protoplasts . The presence of antigens on the PS of the plasma membrane could also be demonstrated on micrographs by the indirect ferritin-antibody labeling technique followed by freeze-etching and shadowing of the membranes.

Biochim Biophys Acta, 1980 Nov 6, 616(1), 82 - 8
Role of cations in the regulation of baker's yeast AMP deaminase; Yoshino M et al.; The effect of polyamines and divalent cations including alkaline earth metals and transition metals on the AMP deaminase (AMP aminohydrolase EC 3.5.4.6) purified from baker's yeast was investigated . (1) Polyamines and alkaline earth metals activated the enzyme in the absence of ATP: these cations largely enhanced the maximal velocity without alteration of S0.5 and nH (Hill coefficient) values . However, transition metals acted as potent inhibitors, which decreased the maximal velocity of the enzyme in the absence of ATP . (2) All of the divalent cations showed an activation of the enzyme in the presence of ATP, followed by a progressive decrease in activity as the concentrations of transition metals increased . (3) The increase in the concentrations of polyamines or alkaline earth metals showed no more activating effect when the enzyme was fully activated by the addition of excess alkali metals in the absence of ATP, but divalent cation-activation was observed in the presence of ATP even if alkali metals were saturating . These results suggest the presence of two types of binding sites for cations: 1, the sites for free cations and 2, those for ATP-metal complexes . The former sites include the activating sites for alkali metals, polyamines and free alkaline earth metals, and the inhibitory sites for free transition metals . The latter sites are the activating sites for ATP-metal complexes, which are suggested to be commonly occupied by alkaline earth metals and transition metals and to form an ATP bridge (E-ATP-M) complex.

Experientia, 1980 Oct 15, 36(10), 1153 - 4
Inhibition of thiamine transport in baker's yeast by methylene blue; Iwashima A et al.; Methylene blue was found to inhibit thiamine transport competitively (Ki = 0.63 microM) in baker's yeast . The dye was also effective in abolishing the growth inhibition of Saccharomyces cerevisiae by pyrithiamine which is known to be taken up by a common transport system for thiamine in yeast cells . A possible mechanism for the inhibition by methylene blue of the thiamine transport system in baker's yeast is discussed.

Biochim Biophys Acta, 1980 Oct 3, 592(3), 415 - 30
Studies on the structure and conformation of yeast mitochondrial ATPase using aurovertin and methanol as probes; Stutterheim E et al.; 1 . The isolation of the mitochondrial ATPase F1 and its beta-subunit from commercial baker's yeast (Saccharomyces cerevisiae) is described . 2 . The molecular weight determined by ultracentrifugation is 340000 +/- 30000 . Gel chromatography indicates a molecular weight of 300000 +/- 20000 . 3 . Fluorimetric titration of the isolated enzyme with aurovertin reveals two binding sites per molecule . The isolated beta-subunit binds aurovertin in a 1 : 1 stoicheiometry . It is concluded that the ATPase molecule contains two aurovertin-binding beta-subunits . 4 . The stabilizing agent methanol influences both the measured Kd and the concentration of binding sites for aurovertin . These results fit a model in which both F1 and aurovertin are distributed between aqueous and methanol phases . 5 . The effect of methanol on the ATPase activity can be described in terms of the model proposed by Recktenwald and Hess (Recktenwald, D . and Hess, B . (1977) FEBS Lett . 76, 25-28) . It is proposed that methanol enhances the affinity of the regulatory site for ATP, but at higher concentrations prevents the interaction between the regulatory and catalytic sites . 6 . Since HSO(-3), a typical effector of the assumed regulatory site of F1, has no effect on the binding of aurovertin, it is concluded that the binding site of aurovertin is not correlated with the regulatory site . 7 . The inhibition of ATPase activity by aurovertin is slowly (t 1/2 = 70 s) induced during turnover conditions . 8 . From the effect of methanol on the inhibition of ATPase activity by aurovertin it is concluded that under turnover conditions the conformation is such that the aurovertin-binding sites have a 6-fold higher affinity for methanol than under resting conditions.

Antibiotiki, 1980 Oct, 25(10), 735 - 8
{Use of enzymatic hydrolysates of microorganism biomass in media for culturing Str . lactis, the producer of nisin}; Baranova IP et al.; Fermentative hydrolysates (FH) of the protein-vitamin complex (PVC) were used for the first time in media for cultivation of Str . lactis . It was found that the FH may be used in the media for cultivation of streptococci as a source of nitrogen instead of the yeast autolysate from baker's yeast and the level of nisin biosynthesis did not change . The replacement of the yeast autolysate by the FH is possible in both flasks with low volumes of the medium and 401 fermenters with 25 liters of the medium . The BVC FH is not a food raw material and its cost is low, which makes it advantageous.

Biochim Biophys Acta, 1980 Aug 7, 614(2), 367 - 72
Inhibition of UDP-N-acetylglucosamine pyrophosphorylase by uridine; Yamamoto K et al.; UDP-N-acetylglucosamine pyrophosphorylases (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.23) from baker's yeast and Neurospora crassa IFO 6178 were inhibited by uridine which is the nucleoside moiety of UDP-GlcNAc . The inhibition was shown in both directions of pyrophosphorolysis and of synthesis of UDP-GlcNAc . Kinetic analysis revealed that uridine demonstrated a noncompetitive type of inhibition with UDP-GlcNAc and competitive inhibition with PPi . The Ki values for the baker's yeast enzyme were 1.8 mM for UDP-GlcNAc and 0.16 mM for PPi, and the values for the Neurospora enzyme were 1.1 mM for UDP-GlcNAc and 0.15 mM for PPi, respectively . Uridine did not bind irreversibly to the enzyme, as the activity was restored with dialysis . No other nucleosides caused inhibition of the enzyme activity except uridine . Some uridine derivatives, such as 5-hydroxyuridine, 5,6-dihydrouridine and pseudouridine, also inhibited the enzyme activity . But doexyuridine showed only slight inhibition, and 5'-UMP and orotidine caused no inhibition of the enzyme activity.

Eur J Biochem, 1980 Aug, 109(1), 39 - 49
Purification and characterization of two high-affinity (adenosine 3',5'-monophosphate)-binding proteins from yeast . Identification as multiple forms of glyceraldehyde-3-phosphate dehydrogenase; Brownlee AG et al.; Two high-affinity cAMP-binding proteins (I and II) have been purified to homogeneity from baker's yeast by a procedure avoiding proteolytic damage . These proteins have been identified as multiple forms of glyceraldehyde-3-phosphate dehydrogenase . The two cAMP-binding proteins are similar in affinities for cAMP, have identical elution volumes on gel filtration, and contain one type of subunit (Mr 37 500) . The form II of glyceraldehyde-3-phosphate dehydrogenase is free of NAD+ and has a Kd of 1.3 X 10(-6) M with respect to cAMP . A marked concentration-dependent self-association of the subunits of the form-II protein was revealed by Yphantis sedimentation equilibrium studies . Significant monomer concentrations are present at total concentrations less than 0.02 mg/ml . Conventional sedimentation equilibrium analyses indicated a tetramer Mr of 170 000 . The high-affinity binding of cAMP to glyceraldehyde-3-phosphate dehydrogenase may significantly reduce intracellular cAMP levels and is also discussed in relation to the nature of eukaryote cAMP-binding proteins with similar native or subunit Mr values which are at present functionally undefined.

Prikl Biokhim Mikrobiol, 1980 Jul-Aug, 16(4), 578 - 83
{Isolation and characterization of microorganisms with mannanase activity}; Pavlova IN et al.; Twenty-three strains of soil microorganisms synthetizing extracellular mannanase were isolated on the medium containing baker's yeast mannan as the sole carbon source . Ten most active strains were identified with respect to their genera . These microorganisms proved capable to hydrolyze mannans, whose mannopyranose units were bound by alpha-1,2-and alpha-1,6-bonds . In addition, 5 of these strains were able to hydrolyze mannans whose mannose residues were bound by alpha-1,3-bond, and 2 strains could synthetize mannanase active towards yeast beta-mannane with 1,3 and 1,4 bonds . The strains tested showed a very low, if any, lytic activity towards yeast Saccharomyces and Candida . This gives evidence that mannanase is an enzyme unable to perform independently lysis of the yeast cell wall.

Prikl Biokhim Mikrobiol, 1980 Jul-Aug, 16(4), 517 - 22
{Influence of oxygen saturation of Saccharomyces cerevisiae suspension in a magnetic field on yeast activity}; Mazur PIa; The suspension of baker's yeast Saccharomyces cerevisiae, race 14, was exposed to a magnetic field, oxygen saturation, and oxygen saturation in the magnetic field . It was found that saturation of the yeast suspension passed at a rate of 1--3 m/min through a magnetic field of 3.10(5)--4.10(5) A/m with molecular oxygen at a pressure of 0.1 MPa to reach a concentration of 16--20 mg/l resulted increases of the yeast rising force, gas formation, 1.5--2.0-fold fermentation rate, 3.7-fold maltase activity, and 3-fold dough viscosity as compared to the control.

J Cell Biol, 1980 Jul, 86(1), 113 - 22
Prefracture and cold-fracture images of yeast plasma membranes; Steere RL et al.; Fracture-temperature related differences in the ultrastructure of plasmalemma P faces of freeze-fractured baker's yeast (Saccharomyces cerevisiae) have been observed in high-resolution replicas prepared in freeze-etch systems pumped to 2 X 10(-7) torr in which the specimens were protected from contamination by use of liquid nitrogen-cooled shrouds . Two major P-face images were observed regardless of the source of the yeast, the age of the culture, the growth temperature, the physiological condition, or the suspending medium used: (a) a "cold-fracture image" with many strands closely associuated with tubelike particles (essentially the same image as those previously published for yeast freeze-fractured at 77 degrees K), and (b) a "prefracture image" characterized by the presence of more distinct tubelike particles with few or no associated strands (for aging cultures, the image recently referred to as "paracrystalline arrays" of "craterlike particles") . Both types of P-face image can be found in separate areas of single replicas and occasionally even within a single plasma membrane . Whereas portions of replicas known to be fractured at any temperature colder than 218 degrees K reveal only the cold-fracture image, prefracture images are found in cells intentionally fractured at 243 degrees K and in cracks or fissures which develop during the freezing of other specimens . These findings demonstrate that the prefracture image results from the fracturing of specimens at some temperature above 230 degrees K, no t from fracturing specimens at some temperature between 173 degrees and 77 degrees K, and not from the use of "starved" yeast cells.

Biochim Biophys Acta, 1980 Jun 13, 613(2), 318 - 23
delta 1-Pyrroline-5-carboxylate reductase from Baker's yeast . Purification, properties and its application in the assays of L-delta 1-pyrroline-5-carboxylate and L-ornithine in tissue; Matsuzawa T et al.; delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) from Baker's yeast has been purified and characterized . Purification to an apparently homogenous protein was effected by using 'reagent-grade' water containing dithiothreitol and by maintaining a constant pH 7.5, because of the instability of the enzyme protein . The enzyme was purified approximately 200-fold from the crude extract of baker's yeast, and it is a negatively-charged protein with a molecular weight of 125 000, containing an active SH group which participates in binding with NAD(P)H . The Km value for DL-pyrroline-5-carboxylate is 0.8 x 10(-4) M; for NADH, it is 4.8 x 10(-5) M; and for NADPH, it is 5.6 x 10(-5) M . These Km values are much smaller than those of enzymes from other sources . The purified enzyme is free of contaminating enzymes which might interfere with its use in assays . The enzyme has been applied successfully to the assays of L-delta 1-pyrroline-5-carboxylate and L-ornithine in tissue, and in vivo levels of these amino acids in rat liver are reported.

Eur J Biochem, 1980 May, 106(1), 151 - 9
Flavocytochrome b2 from baker's yeast . Computer-simulation studies of a new scheme for intramolecular electron transfer; Pompon D; It has been shown recently that the use of L-(+)-{2-2H}lactate as substrate instead of unlabeled L-(+)-lactate induces a lowering of the flavin reduction rate of cytochrome b2 by a factor of 8 {D . Pompon, M . Iwatsubo, and F . Lederer (1980) Eur . J . Biochem . 104, 479--488} . This high isotope effect enabled us to study electron transfer between prosthetic groups at a very low rate of electron entry . The kinetic scheme for electron transfer in cytochrome b2 proposed by Capeillere-Blandin {Eur . J . Biochem . 56, 91--101 (1975)} is examined here in the light of the kinetic data reported in our preceding paper . This study indicates some disagreements, particularly at a low rate of electron entry . New kinetic schemes capable of explaining data obtained with deuterated lactate are proposed . These new schemes differ from that of Capeillere-Blandin in that: (a) the hypothesis of simultaneous prosthetic group reduction for the two protomers of one given dimer is abandoned; (b) the limiting step of the slow phases of heme and flavin reduction is a slow interprotomer electron exchange between a heme pair, a flavin pair or heme and flavin; (c) a rather fast conformational change controlled by the redox state of heme or flavin of one promoter can modulate the rate of electron transfer in another promoter . These new kinetic schemes allow us to determine the rate of intraprotomer and interprotomer electron transfer and to decide precisely how these steps are modified by the proteolytic cleavage of 'intact' enzyme to 'cleaved' enzyme.

J Biochem (Tokyo), 1980 May, 87(5), 1321 - 6
Purification and characterization of yeast protease B; Fujishiro K et al.; Protease B {EC 3.4.22.9} was purified from baker's yeast by plasmolysis of yeast, acid activation, acid precipitation, and column chromatographies on QAE-Sephadex, SP-Sephadex, D-tryptophan methyl ester-Sepharose 4B and Sephadex G-100 . The purified enzyme was inhibited by phenylmethylsulfonyl fluoride and sulfhydryl-blocking reagents . Chymostatin and antipain at extremely low concentrations (1 micro M) inhibited the protease B . The effects of the enzyme on various yeast enzymes were examined by measuring their inactivation . The enzyme inactivated 6-phosphogluconate dehydrogenase {EC 1.1.1.44} and uricase {EC 1.7.3.3}, but not malate dehydrogenase {EC 1.1.1.37}, alcohol dehydrogenase {EC 1.1.1.1}, glutamate dehydrogenase {EC 1.4.1.3}, glucose-6-phosphate dehydrogenase {EC 1.1.1.49} or hexokinase {EC 2.7.1.1}.

Biochemistry, 1980 Apr 15, 19(8), 1676 - 80
A novel enzymatic activity of phenylalanyl transfer ribonucleic acid synthetase from baker's yeast: zinc ion induced transfer ribonucleic acid independent hydrolysis of adenosine triphosphate; Igloi GL et al.; Phenylalanyl-tRNA synthetase from baker's yeast in the presence of phenylalanine or other amino acids misactivated by the enzyme, ATP, and low concentrations of Zn2+ is able to hydrolyze ATP to AMP and PPi very efficiently . After dialysis of the enzyme against ethylenediaminetetraacetic acid (EDTA), this amino acid dependent but tRNAPhe-independent hydrolysis is suppressed to negligible levels . The ATP hydrolysis can be restored by the addition of Zn2+ to the EDTA-dialyzed enzyme . During aminoacylation of tRNAPhe the Zn2+-induced ATP hydrolysis parallels the aminoacylation reaction, leading to nonstoichiometric production of AMP . Mechanistically, we conclude that Zn2+ can be bound to phenylalanyl-tRNA synthetase and can influence the stability of ATP if an activatable amino acid is present . The influence of Zn2+, if any, on the aminoacylation of tRNAPhe is not known . In practice, this side reaction is of the utmost importance in all cases in which the fate of ATP during aminoacylation is followed, especially if the stoichiometry of ATP consumption in relation to Phe-tRNAPhe formation has to be determined.

Eur J Biochem, 1980 Apr, 105(2), 343 - 51
Bromopyruvate as an affinity label for baker's yeast flavocytochrome b2 . Stoichiometry of incorporation and localization on the peptide chain; Alliel PM et al.; We have reported in a previous communication a kinetic study showing bromopyruvate to behave as an active-site-directed reagent for flavocytochrome b2 . It is shown here that inactivation is accompanied by incorporation of 3 mol reagent/subunit of oxidized intact enzyme and 4 mol reagent/subunit nicked enzyme . Only one of the modifications is presumed to be responsible for activity loss . All labeled groups are found to be cysteines . Incubation of reduced nicked enzyme with bromopyruvate results in total protection of activity and loss of only one sulfhydryl group . A subsequent incubation in the presence of the competitive inhibitor sulfite leads to some more loss of non-essential groups . After these two pretreatments, incubation in the presence of bromo{2-14C}pyruvate results in incorporation of 1.2--1.5 mol reagent/subunit concomitant with the loss of about 0.8 active site . A study of the distribution of label between fragments alpha and beta has been carried out using gel electrophoresis and Sephadex filtration after selective proteolysis . It is shown that the active-site sulfhydryl group corresponds to one of the four cysteines situated in the last two thirds of fragment alpha . The structural and functional implications of these results is discussed.

Biochemistry, 1980 Mar 18, 19(6), 1172 - 6
Evidence that deoxyribonucleic acid photolyase from baker's yeast is a flavoprotein; Iwatsuki N et al.; DNA photolyase purified from baker's yeast by affinity chromatography on UV-irradiated DNA noncovalently bound to cellulose and by chromatography on activated thiol-Sepharose 4B yields a single protein band having a molecular weight of 51 000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The molecular weight, 53 000, determined by gel filtration was in good agreement . Upon denaturation of photolyase by heat or 8 M urea, flavin adenine dinucleotide (oxidized) was isolated from the mixture and identified by thin-layer chromatography and spectral analysis . In contrast to flavoproteins to which flavin adenine dinucleotide (oxidized) is bound which generally exhibit two absorbance maxima between 300 and 500 nm, photolyase has only one at 380 nm . These findings and the similar characteristics of the absorbance and emission spectra of native photolyase with those of flavoproteins in which the chromophore is considered to be the 4a,5-reduced flavin have led us to propose this configuration for the photolyase chromophore . The difference in properties of yeast photolyase compared to the one reported previously supports the idea that there are two photolyases in baker's yeast.

J Biochem (Tokyo), 1980 Mar, 87(3), 841 - 6
Purification and enzymatic properties of glyoxylate reductase II from baker's yeast; Fukuda H et al.; Glyoxylate reductase II was purified about 2,400-fold from a cell extract of baker's yeast by protamine sulfate treatment, and column chromatographies on DEAE-cellulose, hydroxylapatite, Sephadex G-150, and phosphocellulose . The purified enzyme was electrophoretically homogeneous . The molecular weight was determined to be approximately 65,000 by gel filtration . The enzyme was greatly stabilized by the addition of 20% (v/v) glycerol . It catalyzed the reduction of glyoxylate and hydroxypyruvate and was specific for NADPH as an electron donor, but showed slight affinity towards NADH . The Michaelis constants for glyoxylate, hydroxypyruvate, NADPH, and NADH were found to be 16mM, 1.4 mM, 5.7 microM, and 0.43 mM, respectively . The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and iodoacetate, but inhibition was prevented by dithiothreitol (DTT) or L-cysteine . The reduction of glyoxylate and hydroxypyruvate was not stimulated by anions.

Eur J Biochem, 1980 Mar, 104(2), 479 - 88
Flavocytochrome b2 (Baker's yeast) . Deuterium isotope effect studied by rapid-kinetic methods as a probe for the mechanism of electron transfer; Pompon D et al.; The use of DL-{2-2H}lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30 degrees C has previously yielded an isotope effect of 5 {F . Lederer (1974) Eur . J . Biochem . 46, 393--399} . We report here studies carried out at 5 degrees C with L-{2-2H}lactate, where flavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor . The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates . It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state . Consequently, two slightly rate-limiting steps occur after the first one where the alpha-hydrogen is abstracted . It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme . The studies were carried out in parallel on intact and cleaved cytochrome b2 . The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps . Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer {Capeillere-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur . J . Biochem . 54, 549--566} . An alternative model based on computer-simulation studies will be presented in a paper in this journal.

Eur J Biochem, 1980 Mar, 105(1), 85 - 92
ATP:AMP phosphotransferase from baker's yeast . Purification and properties; Ito Y et al.; An improved homogeneous preparation of adenylate kinase (ATP:AMP phosphotransferase, ATP + AMP in equilibrium 2 ADP) from baker's yeast was attained by extraction using ethyl acetate and successive column chromatography on Affi-Gel blue, Sephadex G-100, phosphocellulose and Sephacryl S-200 . The overall purification is about 670-fold with a yield of 23% and final specific activity of 1900 units/mg protein . The enzyme preparation is a single band in isoelectrofocusing with a pI of 5.7 . By sodium dodecyl sulfate gel electrophoresis and gel chromatography the molecular weight is 27 500 . Among the nucleoside monophosphates investigated (AMP, CMP, GMP, IMP and UMP) only AMP reacts with ATP (dATP) . ATP (dATP) is about one order of magnitude more active than CTP, GTP, ITP and UTP . The enzyme catalyzes only in the presence of a divalent metal cation, namely Mg2+, Ca2+, Co2+, Mn2+ and Ni2+, and the reaction rate is maximal at about 0.5 M NaCl . The binding of the substrates also takes place in the absence of metal . The dissociation constants for ATP, MgATP, CTP, GTP, UTP and AMP are 3.4, 4.2, 18.5, 17.2, 23.8 and 39.3 microM respectively . The amino acid composition of the purified enzyme is: 32 aspartic acid + asparagine, 12 threonine, 12 serine, 27 glutamic acid + glutamine, 16 proline, 21 glycine, 24 alanine, 11 valine, 9 methionine, 17 isoleucine, 25 leucine, 4 tyrosine, 7 phenylalanine, 2 half-cystine (no free sulfhydryl), 23 lysine, 6 histidine, 10 arginine and 2 tryptophan, totalling 260 residues.

J Biol Chem, 1980 Feb 10, 255(3), 937 - 42
The inactivation of saccharopine dehydrogenase (L-lysine-forming) by diethyl pyrocarbonate; Fujioka M et al.; Saccharopine dehydrogenase (epsilon-N-(L-glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine-forming) EC 1.5.1.7) from baker's yeast is inactivated by diethyl pyrocarbonate . Spectrophotometric studies show that the inactivation results from the modification of 3 histidyl residues/molecule of enzyme . The sulfhydryl content of the enzyme is unchanged by modification . The reversibility of inactivation by hydroxylamine and the pH dependence of inactivation are also consistent with the inactivation being due to modification of the histidyl residue . Although the coenzyme and substrates are without effect when added singly, the inactivation is completely protected by alpha-ketoglutarate in the presence of a saturating concentration of NADH . Since alpha-ketoglutarate binds only to the enzyme . NADH complex, the results suggest that the inactivation is due to modification of the residue at or near the substrate-binding site . Under the conditions where the inactivation is largely protected by NADH plus alpha-ketoglutarate, 2 histidyl residues appear to be modified suggesting that only 1 residue involved in the catalytic activity . The modification appears to prevent the binding of alpha-ketoglutarate, but not of the coenzyme, to the enzyme . The protein fluorescence of the native and modified enzymes is quenched by NAD+ and NADH . However, the NADH titration curve of the modified enzyme is not affected by alpha-ketoglutarate, in contrast to the native enzyme which shows an increase in the apparent affinity for the coenzyme in the presence of alpha-ketoglutarate.

Folia Microbiol (Praha), 1980, 25(6), 501 - 4
Preparation of zymosan from yeast cell walls; Holan Z et al.; Cell walls of the yeast Saccharomyces cerevisiae after disintegration and protoplasm removal by centrifugation and repeated washing were suspended in 0.5 M Na2HPO4, pH 7.8-8.0, as a 5% or 10% suspension, depending on the mode of heating . The suspension was boiled for 3 h, purified by repeated washing with water and ethanol and dried . The yield was approximately 1.8% of the starting amount of pressed commercial baker's yeast.

Folia Microbiol (Praha), 1980, 25(4), 311 - 7
Extrusion of metabolites from baker's yeast during glucose-induced acidification; Sigler K et al.; Extrusion of metabolites (glycerol, lactic, malic, and succinic acid) during the medium acidification caused by resting baker's yeast supplied with 200 mM glucose was studied under aerobic and anaerobic conditions and in the absence and presence of 14 mM KCl . The maximum levels of glycerol and of the sum of acids (about 13 and 8 mM, respectively) were attained anaerobically; aerobiosis reduced the levels by 40-50% and the presence of K+ ions by another 10-20% . The time courses of glucose consumption and medium acidification were similar aerobically and anaerobically . The glucose consumption curves exhibited a short plateau about 2 min after glucose addition, caused probably by a rapid osmotic equilibration of glucose across the cell mambrane . Metabolite extrusion indicates that at high glucose concentrations the alcohol dehydrogenase reaction is supplemented by other reactions aiding in the maintenance of a balanced NAD+/NADH ratio in the cells.

Folia Microbiol (Praha), 1980, 25(4), 306 - 10
Gas chromatographic determination of extracellular metabolites produced by baker's yeast during glucose-induced acidification; Wurst M et al.; Gas chromatographic separation of metabolites extruded during the medium acidification by resting baker's yeast supplied with glucose is described . Silylation with bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane and a direct chromatography on Chromosorb G-AW-DMCS with 2.5% SE-52 (lactic, pyruvic, succinic, glyceric, fumaric, malic), polyols (glycerol, arabinitol) and sugars (glucose) . The yeast was found to extrude glycerol, lactic, malic, and succinic acid.

Eur J Biochem, 1980, 107(1), 47 - 50
Phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases . Substrate specificity in the ATP/PPi exchange with regard to ATP analogs; Freist W et al.; The analogs of ATP have been tested in the ATP/PPi exchange reaction of phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases from baker's yeast . Three compounds are substrates for phenylalanyl-tRNA, seven for lysyl-tRNA, two for isoleucyl-tRNA and five for arginyl-tRNA synthetase . Their Km and V values have been determined . No analog was an inhibitor . (3'-dATP), 3'-Deoxyadenosine 5'-triphosphate which is an inhibitor of the four enzymes in the aminoacylation reaction, becomes a good substrate in the PPi exchange . Additionally lysyl-tRNA synthetase accepts two analogs with modifications at position 6 of the purine and three analogs modified at the ribose moiety as substrates in the PPi exchange, whereas these compounds are inactive or inhibitors in the aminoacylation reaction . In general the enzymes are less specific in the ATP/PPi exchange and the results indicate a more sophisticated proof of the nucleotide moiety upon aminoacylation . This could occur with the aminoacyladenylate intermediate as well as with any other intermediate.

Cytogenet Cell Genet, 1980, 26(1), 36 - 40
Yeast stimulation of bone marrow mitosis for cytogenetic investigations; Lee MR et al.; We report a simple, dependable method for stimulating bone marrow mitosis in small mammals . Subcutaneous injections of a suspension of active baker's yeast may elevate the mitotic index as much as six times or more . Additionally, the metaphases obtained are easily spread when air dried, and the chromosomes are readily banded . This method should prove useful to investigators who wish to use bone marrow as a source of chromosomes for cytotaxonomic studies or for studies of specific chromosome damage in vivo.

Hoppe Seylers Z Physiol Chem, 1980 Jan, 361(1), 61 - 8
Mild purification procedure and subunit structure of glucosephosphate isomerase from baker's yeast; Tamaki N et al.; A mild procedure for the purification of glucosephosphate isomerase from baker's yeast (Saccharomyces cerevisiae) is reported . The purified enzyme was homogeneous and did not contain charge isomers as shown by polyacrylamide gel electrophoresis as well as DEAE-Sepharose column chromatography . Its molecular weight determined by gel filtration and sucrose density gradient centrifugation was approximately 120 000, which agreed with that of the enzyme in the crude extract as well as that of the renatured enzyme . Gel filtration in 6M guanidine/HCl as well as acrylamide gel electrophoresis of sodium dodecyl sulfate denatured glucosephosphate isomerase showed one single peak and gave a subunit molecular weight of 60 000 . Cross-linking patterns obtained with yeast glucosephosphate isomerase after treatment with dimethyl suberimidate resulted in the appearance of dimers as the largest-linked product of the enzyme subunit . After dissociation the enzyme can readily be reassociated and renatured with a yield of maximum 73% and a pseudo first order rate constant of 0.12 min-1 at 25 degrees C.

Arch Exp Veterinarmed, 1980, 34(3), 353 - 6
{Effect of inorganic and yeast-borne tin on rats}; Pfaff K et al.; Tin incorporated in baker's yeast was applied to male Wistar rats over 21 days . The doses corresponded to 1/500 or 1/50 of LD50 of SnCl2 x 2H2O and 1/50 of LD50 of SnCl2 x 2H2O . The effects on activities of certain selected enzymes as well as on tin levels in liver and kidneys were tested . Some of the enzymes tested were found to reflect even exposures much below the point recordable from accumulation of the metal . Serum lactate-dehydrogenase is recommended as a sensitive indicator enzyme in the context of tin exposure.

Nahrung, 1980, 24(4-5), 455 - 61
{Effects of tin on rats . II . Comparison of the effects of tin (II) chloride and yeast-incorporated tin}; Kujawa M et al.; After application of SnCl2 and tin incorporated into baker's yeast, the effects on carbonic anhydrase (CA), glutathione peroxidase (GPx), lactate dehydrogenase (LDH), alkaline phosphatase (AP) and leucine aminopeptidase (LAP) were measured . The tin contents of liver and kidneys were determined . CA and GPx are not affected . AP and LAP are inhibited by high concentrations of tin (as SnCl2) . Tin incorporated into yeast exerts no effect . Inorganic tin produces increases in liver and kidneys.

J Mol Evol, 1979 Dec, 14(4), 251 - 8
Compositional relatedness of aldehyde reductases from several species; Davidson WS et al.; The amino acid compositions of several monomeric NADPH-dependent aldehyde reductases from a variety of species have been determined and analyzed by the difference index method of Metzger et al . (1968) . The difference indexes among mammals range from 4.15 - 6.10 indicating considerable homology . Comparison of chicken aldehyde reductase with mammalian aldehyde reductases gave values in the range 6.8 - 9.9 suggesting a close relationship whereas the difference indexes for the enzymes from fruit fly and Baker's yeast versus vertebrate aldehyde reductases (10.9 - 14.4) indicate more distant relationships . The extent of sequence homology among aldehyde reductases from these species was estimated from a plot of difference index versus percent sequence difference for oxido-reductases of known sequence . From this plot, and using a mammal-chicken divergence time of 300 million years and a mammalian order split of 75 million years, the rate of evolution of aldehyde reductases was calculated to lie in the range 5.8 - 15.6% sequence difference per 100 million years . Comparison with rates of evolution of oligomeric dehydrogenases indicates that aldehyde reductases comprise the most rapidly evolving family of oxido-reductases . This is probably related to the monomericity of aldehyde reductases since there is a direct correlation between the number of subunits and the rate of evolution.

Prikl Biokhim Mikrobiol, 1979 Nov-Dec, 15(6), 822 - 6
{Dehydration effect on the fatty acid composition of lipids in baker's yeast Saccharomyces cerevisiae 14}; Auzinia LP et al.; The effect of dehydration on the fatty acid composition of lipids in baker's yeast Saccharomyces cerevisiae 14 cultivated on the ethanol (1.5%) containing medium in the presence or absence of glutamic acid and biotin or on molasses (2%) was studied . In spite of a relatively low drying temperature (35 degrees C), the correlation between saturated and non-saturated fatty acids may vary within 40--60 min.

J Biochem (Tokyo), 1979 Nov, 86(5), 1331 - 6
AMP deaminase from baker's yeast . Kinetic and molecular properties; Murakami K; The kinetic and molecular properties of AMP deaminase {AMP aminohydrolase, EC 3.5.4.6} purified from baker's yeast (saccharomyces cerevisiae) were investigated . The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner . Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent . Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates . Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule . The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies . On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure . Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.

Biochim Biophys Acta, 1979 Sep 28, 574(3), 448 - 60
Utilization of endogenous diacylglycerol for the synthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine by lipid particles from baker's yeast (Saccharomyces cerevisiae); Christiansen K; The activity of the enzymes diacylglycerol acyltransferase (EC 2.3.1.20), cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) have been measured in a lipid particle preparation from baker's yeast (Saccharomyces cerevisiae) with endogenous 1,2-diacylglycerol as substrate . For all three enzymes the rate of diacylglycerol utilization was established with respect to substrate and Mg2+ concentration . Neither of the enzyme activities was stimulated significantly by addition of diacylglycerols . The conversion of diacylglycerol into triacylglycerol in the presence of CDP-choline and CDPethanolamine, and the synthesis of phospholipids in the presence of acyl-CoA either added or generated in situ were studied . Neither CDPcholine nor CDPethanolamine had an effect on triacylglycerol synthesis . Exogenous acyl-CoA had no effect on either choline- or ethanolaminephosphotransferase activity . However, when the necessary substrates for formation of acyl-CoAs in situ (ATP, CoA, Mg2+ and free fatty acids) were added a decrease in both cholinephosphotransferase and ethanolaminephosphotransferase activity was observed . This inhibition was shown to be due to ATP and might explained as a result of chelation of the Mg2+, a necessary activator of both the choline- and the ethanolaminephosphotransferase.

Biochim Biophys Acta, 1979 Sep 12, 570(1), 157 - 66
AMP deaminase from baker's yeast . Purification and some regulatory properties; Yoshino M et al.; AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP . The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals . ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM . P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration . In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction . AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells . These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.

Biochem J, 1979 Sep 1, 181(3), 539 - 43
The temperature-dependence of adenylate cyclase from baker's yeast; Londesborough J et al.; The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol) . Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol . The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism.

Prikl Biokhim Mikrobiol, 1979 Jul-Aug, 15(4), 605 - 11
{Comparison of different methods for measuring nucleic acids and protein in the biomass, half-products and preparations of E . coli and yeast}; Pupkova VI et al.; In the E . coli biomass, half-products and RNA preparations a comparative measurement of nucleic acids and protein was carried out using different methods . As a result, the following methods can be recommended: to assay RNA in the biomass--spectrophotometry, to determine RNA in half-products and preparations--the phosphate method, to assess DNA--the diphenyl amine method, and to measure protein--according to Lowry . The same methods can be recommended to estimate RNA from baker's yeast.

J Biochem (Tokyo), 1979 Jul, 86(1), 105 - 10
Purification and properties of glyoxylate reductase I from baker's yeast; Tochikura T et al.; The purification and properties of NADPH-linked glyoxylate reductase {EC 1 . 1 . 1 . 79} from baker's yeast were studied . Two active fractions (peak I and peak II) were isolated by DEAE-cellulose column chromatography . The peak I fraction was purified to homogeneity by the criteria of disc gel electrophoresis and tentatively designated glyoxylate reductase I . Its molecular weight was calculated to be 31,000 from gel filtration measurements . The enzyme reduced glyoxylate 7 times faster than hydroxypyruvate and was specific for NADPH . The enzyme showed optimum activity between pH 5.5 and 7.2 . The Michaelis constants for glyoxylate and NADPH were found to be 13 mM and 4 microM, respectively . The enzymic activity was not significantly affected by anions, except for nitrate and iodide, which were inhibitory.

J Biochem (Tokyo), 1979 May, 85(5), 1165 - 72
Formaldehyde dehydrogenase from Pseudomonas putida . Purification and some properties; Ando M et al.; Formaldehyde dehydrogenase was isolated and purified in an overall yield of 12% from cell-free extract of Pseudomonas putida C-83 by chromatographies on columns of DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite . The purified enzyme was homogeneous as judged by disc gel electrophoresis and was most active at pH 7.8 using formaldehyde as a substrate . The enzyme was also active toward acetaldehyde, propionaldehyde, glyoxal, and pyruvaldehyde, though the reaction rates were low . The enzyme was NAD+-linked but did not require the external addition of glutathione, in contrast with the usual formaldehyde dehydrogenase from liver mitochondria, baker's yeast, and some bacteria . The enzyme was markedly inhibited by Ni2+, Pd2+, Hg2+, p-chloromercuribenzoate, and phenylmethanesulfonyl fluoride . The molecular weight of the enzyme was estimated to be 150,000 by the gel filtration method, and analysis by SDS-polyacrylamide gel electrophoresis indicated that the enzyme was composed of two subunit monomers . Kinetic analysis gave Km values of 67 microM for formaldehyde and 56 microM for NAD+, and suggested that the reaction proceeds by a "Ping-pong" mechanism . The enzyme catalyzed the oxidation of formaldehyde accompanied by the stoichiometric reduction of NAD+, but no reverse reaction was observed.

Biochim Biophys Acta, 1979 Apr 12, 567(2), 503 - 10
Chemical and physical properties of the carboxypeptidase Y-inhibitor from Baker's yeast; Matern H et al.; The purification as well as some characteristics of the carboxypeptidase Y-inhibitor from baker's yeast have been described in a previous report (Matern, H., Hoffmann, M . and Holzer, H . (1974) Proc . Natl . Acad . Sci . U.S . 71, 4874-4878) . In this paper, chemical and physical properties of the purified inhibitor are presented . The molecular weight was estimated at 23 400--24 000 and appears to be a monomeric unit . Amino acid analysis and carbohydrate studies are given, showing the existence of three disulfide bonds and one sulfhydryl group per molecule and the absence of carbohydrate residues . The N-terminal amino acid is blocked by an acetyl group . The C-terminal amino acid is lysine . The isoelectric point (pI) is 6.6 and the inhibitor-enzyme complex is stable (at 25 degrees C) betwen pH 5 and 9 . The apparent Ki value was calculated as 2.5.10(-9)M.

Eur J Biochem, 1979 Apr 2, 95(2), 341 - 8
Reversible inactivation of tRNA nucleotidyltransferase from baker's yeast by tRNAPhe containing iodoacetamide-alkylated 2-thiocytidine in normal and additional positions; Kroger M et al.; 2-Thiocytidine 5'-triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase . s2CMP can be incorporated into both cytidine sites of the C-C-A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions . This was shown by alkylation of the 2-thiocytidine residues with iodo{14C}acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs . The incorporation of the 3'-terminal AMP is not influenced by the additional s2CMP residues at pH 9.0 . However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position . Two different tRNAs with terminal 2-thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase . This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2-thiocytidine in the normal position 75 . The addition of 2-mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA . This reaction identifies the attacking nucleophile of the enzyme as cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously . The mechanism of the enzymatic and chemical reactions is discussed.

Immun Infekt, 1979 Feb, 7(1), 21 - 3
{Phagocytosis and nitroblue-tetrazolium reduction . Methodical variation (author's transl)}; Neumann G et al.; A variation of the method of Preisig and Hitzig (18) for the determination of phagocytosis and NBT-reduction of polymorphonuclear granulocytes and monocytes in the peripheral blood is described . Contact with infectious cultures of Candida albicans is avoided by the use of baker's yeast for phagocytosis . Methylgreen is replaced by nuclear fast red for staining . The dye solution can be used over a longer period, the stained slides do not lose their colour, the contrast with the reduced blue NBT is more intense . The application of the method is demonstrated by the results of the test in the family of a boy suffering from CGD.

J Gen Microbiol, 1979 Feb, 110(2), 323 - 32
Uptake of trehalose by Saccharomyces cerevisiae; Kotyk A et al.; Trehalose, a storage sugar of baker's yeast, is known not to be metabolized when added to a cell suspension in water or a growth medium and to support growth only after a lag of about 10 h . However, it was transported into cells by at least two transport systems, the uptake being active, with a pH optimum at 5.5 . There was no stoicheiometry with the shift of protons into cells observed at high trehalose concentrations . Trehalose remained intact in cells and was not appreciably lost to a trehalose-free medium . The uptake systems were present directly after growth on glucose, then decayed with a half-life of about 25 min but could be reactivated by aerobic incubation with trehalose, maltose, alpha-methyl-D-glucoside, glucose or ethanol . The uptake systems thus induced were different as revealed by competition experiments . At least one of the systems for trehalose uptake showed cooperative kinetics . Comparative anaysis with other disaccharides indicated the existence in Saccharomyces cerevisiae, after induction with trehalose, of at least four systems for the uptake of alpha-methyl-D-glucoside, four systems for maltose, together with the two for trehalose, variously shared by the sugars, the total of alpha-glucoside-transporting systems being five.

Eur J Biochem, 1979 Jan 2, 93(1), 79 - 94
Yeast tRNA Leu UAG . Purification, properties and determination of the nucleotide sequence by radioactive derivative methods; Randerath E et al.; A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration . Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu) . The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods . Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis . Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods . Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis . 2'-O-Methylated guanosine was characterized using the {gamma-32P}ATP/polynucleotide kinase reaction . The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA . A total of about 20 A260 units of the RNA was used for analysis, i.e . considerably less material than required for conventional spectrophotometric analysis . A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule . The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis . The sequence was read off the gel on the basis of cleavage patterns and size of the fragments . While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides . The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units . Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

Mikrobiologiia, 1979 Jan-Feb, 48(1), 153 - 6
{Effect of cell reserve carbohydrates on an increase in the keeping qualities of baker's yeast}; Bocharova NN et al.; Synthesis of reserve carbohydrates (trehalose and metabolically active glycogen) can be intensified in the cells of Saccharomyces cerevisiae by adding glycerol and magnesium or phosphorus salts to the cultural broth . An increase in the content of reserve carbohydrates in the cells increases their preservation after separation from the substrate.

Scand J Immunol, 1979, 9(1), 9 - 14
A simple method to detect complement receptors using baker's yeast: Y C rosettes; Rivero I et al.; A technique is described to identify complement-receptor-bearing cells, using serum-treated baker's yeast as a ligand . The method consists of incubation of heat-killed baker's yeasts with fresh AB normal serum, freezing, thawing, and washing of the particles, followed by mixing with the cells . Serum is required to coat the yeasts for the rosette formation . Experiments designed to establish the serum factors responsible for the attachment of the particles to cells show that heat inactivation, chelating agents, or anti-C3 treatment prevent rosette formation . This is taken as evidence that yeasts (Y) are coated with complement (C) to compose the reagent for the YC rosette technique . The application of this technique to twenty-five normal individuals demonstrated that a mean of 11.6 per 100 lymphocytes (+/- 4.3) form rosettes; absolute number: 275 (+/- 160) rosette-forming lymphocytes per mm3 . Either AB or autologous fresh serum can be used to coat the yeasts . A combined technique for YC plus E rosettes can be performed allowing the identification and enumeration of four populations of lymphocytes: (a) those having receptors for sheep erythrocytes, (b) complement-receptor-bearing lymphocytes, (c) those having both receptors (D lymphocytes), and (d) non-rosette-forming non-phagocytic cells.

Folia Microbiol (Praha), 1979, 24(6), 449 - 54
The estimation of alcohol dehydrogenase activity in aerobic and anaerobic "permeabilised" baker's yeast cells; Israelstam GF; The in vivo and in vitro activity of alcohol dehydrogenase from baker's yeast maintained under aerobic and anaerobic conditions was measured . In vivo measurements were made in cells "permeabilised" with toluene . Michaelis constants (NAD+ as substrate) were found to be almost identical as those reported for purified preparations . In addition the Km of the enzyme from cells incubated under anaerobic conditions was virtually identical to that from cells from aerobic conditions . The activity of the enzyme was found to be greater (in both "permeabilised" cells and extracts) in cells maintained under nitrogen than air . Cells metabolizing glucose in N2 produced greater levels of ethanol than in air and the rate of NAD+ reduction was also found to be greater in N2 than in air . The results indicate that it was feasible to determine rates of this enzyme in vivo and that the difference in activity of alcohol dehydrogenase under N2 and air may conceivably account for differences in rates of glucose utilisation, ethanol production and NAD+ reduction in air and nitrogen.

Acta Biol Med Ger, 1979, 38(8), 1067 - 79
Kinetic modelling of yeast phosphofructokinase; Reuter R et al.; Phosphofructokinase from baker's yeast (Saccharomyces cerevisiae) is an octameric enzyme which exhibits complex allosteric behaviour . In contrast to mammalian phosphofructokinase, the enzyme does not show association-dissociation behaviour . A systematic kinetic investigation at pH 7.2 in dependence on the substrates, fructose 6-phosphate and ATP as well as on the effectors AMP and ADP is presented . The results are interpreted in terms of a structure oriented theoretical model . Because the two state model of Monod, Wyman and Changeux proved to be insufficient for interpretation of the experimental data, it was extended to a four state model in which the basic conformations R and T of the enzyme are split into subconformations R1 and R2 as well as T1 and T2, respectively . It is assumed that fructose 6-phosphate and the adenine nucleotides influence different allosteric equilibria . The model permits a precise quantitative description of the experimental data.

Z Naturforsch {C}, 1979 Jan-Feb, 34(1-2), 20 - 6
Phenylalanyl-tRNA synthetase from baker's yeast: structural organization of the enzyme and its complex with tRNAPhe as determined by X-ray small-angle scattering; Pilz I et al.; The quaternary structure of the phenylalanyl-tRNA synthetase and its complex with tRNAPhe was studied in dilute solutions by small angle X-ray scattering . For the free synthetase the radius of gyration was determined as 5.5 nm, the volume 523 nm3, the maximum diameter 17.5 nm and the molecular weight as 260,000 using an isopotential specific volume of 0.735 . The overall shape could be best approximated by a flat cylinder with dimensions 18.2 nmx11.5 nmx4nm; the loose structure was approximated by building up the cylinder by spheres (diameter 4.2 nm) . The corresponding parameters of the enzyme tRNA complex were the following: radius of gyration 5.9 nm, volume 543 nm3, maximum diameter 21 nm and molecular weight 290,000 . These parameters suggest an 1:1 complex, whereby it must be assumed that the tRNA molecule is attached in the extension of the longer axis . From the difference in the distance distribution functions of the free enzyme and the complex it is evident that we have to assume a change of conformation (contraction) of the enzyme upon the binding of the specific tRNA.

Acta Biol Med Ger, 1979, 38(11-12), 1639 - 41
Indications of pH-induced conformational changes in phosphofructokinase from baker's yeast; Johansson G et al.; The partition of phosphorfructokinase (EC 2.7.1.11) from baker's yeast between the liquid phases of an aqueous biphasic systeem changes drastically in the pH interval 7-8, in contrast to other proteins . This abnormal behaviour is correlated to changes in sedimentation coefficient and binding capacity of the enzyme in this pH region . Since the molecular weight of phosphofructokinase does not change, these findings must reflect conformational changes in the enzyme molecule.

Acta Biol Med Ger, 1979, 38(10), 1399 - 412
{Effect of magnesium ions on the thermostability of inorganic pyrophosphatase from baker's yeast}; Frommel C et al.; The interaction of magnesium ions with inorganic pyrophosphatase from baker's yeast was studied by means of heat denaturation . The heat inactivation of this enzyme is a biphasic process . The velocities in the initial range and in the subsequent slower part of inactivation are diminished with rising Mg2+ concentration in the inactivation assay . A model is proposed which describes this behavior . It is assumed that two enzyme conformations exist in equilibrium whose conversion rates correspond to the inactivation rate in its order of magnitude . The equilibrium is shifted by Mg2+ . The two enzyme species differ in their Mg2+ binding behavior as evidenced by differences in the half-saturation constants and the cooperativity of the binding . The same conclusions are drawn from the fluorimetric measurement of denaturation of inorganic pyrophosphatase . Besides, an additional Mg2+ binding site is demonstrable, the saturation of which obviously leads to stabilisation of part of the enzyme structure without protecting it against loss of enzymatic activity . With the same method the labilizing effect of Zn2+ on the structure of the inorganic pyrophosphatase from baker's yeast was studied.

Folia Microbiol (Praha), 1979, 24(6), 455 - 61
Activation of proteolytic enzymes during autolysis of disintegrated baker's yeast; Behalova B et al.; Disintegration substantially accelerates autolysis of yeast cells . Three proteases (A, B, and C) take part in the autolytic process, protease A being the activator of the other two enzymes . The role of proteases B and C in the process depends on temperature . At 40 degrees C both proteases are active while at 50 degrees C the major role is played by protease C . At 40 degrees C NaCl acts as inhibitor while at 50 degrees C it activates the process.

J Parasitol, 1978 Dec, 64(6), 1024 - 7
Trichostrongylus colubriformis: cultivation of free-living stages; Khan ZI et al.; Successful cultivation of Trichostrongylus colubriformis from hatched first-stage to third-stage larvae was achieved in media containing NCTC 135, chick embryo extract, fetal calf serum, and either lactalbumin hydrolysate or freshly prepared baker's yeast extract . Medium Tc5, containing yeast extract, was the simplest medium with optimal results . Cultured third-stage larvae were able to produce patent infections in guinea pigs.

Biochemistry, 1978 Nov 14, 17(23), 4900 - 7
Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase; Holland MJ et al.; Yeast poly(adenylic acid)-containing messenger ribonucleic acid isolated from two strains of Saccharomyces cerevisiae was fractionated by preparative polyacrylamide gel electrophoresis in the presence of formamide . Three messenger ribonucleic acids, present at high intracellular concentration, were electrophoretically eluted from the polyacrylamide gels and translated in a wheat germ cell-free extract . The in vitro synthesized polypeptides were identified by tryptic peptide analysis . Messenger ribonucleic acids coding for enolase and glyceraldehyde-3-phosphate dehydrogenase were isolated from commercially grown baker's yeast (strain F1), and messenger ribonucleic acid coding for phosphoglycerate kinase was isolated from Saccharomyces cerevisiae (ATCC 24657) . Significant differences in the spectrum of abundant messenger ribonucleic acids isolated from commercially grown baker's yeast (strain F1) and strain 24657 were observed . When both strains were grown under identical conditions, however, the spectrum of messenger ribonucleic acid isolated from the cells is indistinguishable.

Biochim Biophys Acta, 1978 Nov 10, 527(1), 249 - 55
The affinity chromatography of transketolase; Wood T et al.; A number of possible affinity adsorbents for transketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphateglycolaldehydetransferase, EC 2.2.1.1) were prepared . The behaviour of the enzyme from Candida utilis and from Baker's yeast on columns of these and of Blue Sepharose CL-6B was examined, together with the behaviour of the contaminating enzyme, ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) . A procedure for removing bound thiamine pyrophosphate by dialysis against EDTA was developed . The competitive inhibition of transketolase by oxythiamine and neopyrithiamine was measured and the Ki values obtained of 1.4 and 4.3 mM, respectively, were compared with the affinity of adsorbents prepared from these two inhibitors . Adsorbents containing bound thiamine pyrophosphate were relatively ineffective but those containing epoxy-linked neopyrithiamine and D-ribose 5-phosphate adsorbed the enzyme at pH 7.4 and it could be eluted in a specific manner.

J Biol Chem, 1978 Nov 10, 253(21), 7891 - 7
Multiple forms of carboxypeptidase Y from Saccharomyces cerevisiae . Kinetic demonstration of effects of carbohydrate residues on the catalytic mechanism of a glycoenzyme; Margolis HC et al.; In the course of our further investigation of the active site titration of carboxypeptidase Y, using 4-nitrophenyl trimethylacetate, we have found that carboxypeptidase Y can be isolated in different molecular forms . Carboxypeptidase Y obtained from Fleischmann baker's yeast has a molecular weight of 53,000, as compared to 64,000 for an enzyme species isolated from Anheuser-Busch baker's yeast . The amino acid analyses of both enzymes were essentially identical and very similar to those reported by others . However, we have found that the molecular weight difference is due to a variation in carbohydrate content as determined by gas chromatography . When carboxypeptidase Y was isolated from a single source, Anheuser-Busch baker's yeast, we observed a smaller variation in carbohydrate content . In all cases, sugar analyses revealed only mannose and N-acetylglucosamine to be present . The effect of the enzyme's carbohydrate content on the "burst kinetics" of the 4-nitrophenyl trimethylacetate reaction has been examined . In general, the Anheuser-Busch enzyme, containing more carbohydrate than the Fleischmann enzyme, reacts with a larger apparent bimolecular rate constant, kcat/Km . On the other hand, the deacylation rate constant, k3, is affected only slightly.

Mutat Res, 1978 Nov, 58(2-3), 241 - 50
Genetic activity of chemicals in yeast: DNA alterations and mutations induced by alkylating anti-cancer agents; Ruhland A et al.; The simple eukaryotic organism baker's yeast allows demonstration of primary DNA lesions in parallel with measurement of mutagenicity and lethality after treatment with alkylating chemicals . Several anti-cancer drugs formed cross-linked DNA molecules and were genetically active . The mutagenicity and lethality of these drugs varied substantially and were dependent on the function of some processes of DNA dark-repair.

Int J Pept Protein Res, 1978 Nov, 12(5), 233 - 6
Alkaline isomerization of horse and yeast cytochromes C . Spectrophotometric and circular dichroism studies; Looze Y et al.; Spectrophotometric studies of the alkaline isomerization of horse heart and yeast cytochrome c show that the haemoproteins from Saccharomyces cerevisiae differ significantly from the mammalian cytochrome c . Apparent pKa values of 8.41, 8.40 and 8.73 for isol-1-(the methylated and unmethylated forms) and iso-2-cytochrome c respectively, from baker's yeast were determined and compared with the value of 9.40 found for horse heart cytochrome c . The transitions, measured by observing the decrease of the absorbance at 695 nm as the pH increases, have been found to strictly parallel the decrease in amplitude of the negative circular dichroism band centered at 417 nm . This observation gives additional evidence that this negative band is closely related to the ligation of the heme iron by the sulfur atom of methionine 8u for each of the four haemoproteins examined.

Eur J Biochem, 1978 Oct 16, 90(3), 563 - 9
Binding of Cibacron Blue F3GA to flavocytochrome b2 from baker's yeast; Pompon D et al.; 1 . Flavin-free cytochrome b2 has been prepared by rapid Sephadex filtration at acid pH . The method, which yields an apo-enzyme with high reconstitution potential and has several advantages over previously used procedures, is described in detail . 2 . Flavin-free cytochrome b2 thus prepared is retained by blue-dextran-bound Sepharose . It can be eluted by an increase in ionic strength, by dilute ethylene glycol and specifically by low concentrations of FMN . The holoenzyme is not retarded at all . 3 . Both flavin-free and holocytochrome b2 bind Cibacron blue F3GA with appearance of distinct difference spectra . Cibacron blue is an inhibitor for the holoenzyme, it shows mixed type inhibition with respect to lactate . 4 . It is concluded that there are two types of binding sites for Cibacron blue F3GA on flavocytochrome b2 . Both possess ionic and hydrophobic character; one of them, which is the flavin binding site, is only available in the absence of the cofactor . Taken together these results may mean that the enzyme possesses a local flavin-binding structure similar to the 'dinucleotide fold'.

Nucleic Acids Res, 1978 Oct, 5(10), 3743 - 57
Large-scale isolation of a native deoxyribonucleohistone complex from baker's yeast; Franco L et al.; A method for large scale isolation of a native deoxyribonucleohistone complex from yeast is described . Crude chromatin, obtained after disrupting yeast cells at low ionic strength, contains a large amount of lipids, partially due to contaminating membranes . Most of them are removed by a Triton X-100 treatment, followed by step-gradient centrifugation . About 90% of the pellet may be solubilized by mild procedures, the composition of the soluble material being: histone/DNA = 1.0;nonhistone proteins/DNA = 0.55; RNA/DNA = 0.18 . Histones can be obtained with high purity . Micrococcal nuclease digests DNA to yield a series of oligomeric fragments, with an average repeat length of about 160 base pairs . Circular dichroism spectra show that (theta) 270 is reduced by about 30% when compared to pure DNA and that chromosomal proteins are not denatured . These results indicate that the components of the complex conserve the native state.

Biochim Biophys Acta, 1978 Sep 26, 536(1), 212 - 25
Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae); Sievers G; Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm . All derivatives show negative Soret Cotton effects . The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron . The native ferric enzyme is probably pentacoordinated . The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme . The spectrum of Compound I shows no perturbation of the porphyrin symmetry . The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure . After the removal of the heme group no great changes in the secondary structure can be observed.

Hoppe Seylers Z Physiol Chem, 1978 Aug, 359(8), 993 - 7
Molecular weight and hydrodynamic properties of a proteinase A inhibitor from baker's yeast; Meussdoerffer F et al.; The molecular weight of the proteinase A inhibitor IA3 from baker's yeast was determined by different methods . From gel-filtration experiments, a molecular weight of 19 000 was calculated for the native inhibitor, while under denaturing conditions a molecular weight of 7400 was found . From electrophoretic experiments with the native protein, a molecular weight of 9000 was calculated . A similar value was obtained from the analytical ultracentrifuge, even at a protein concentration of 12 mg/ml . The diffusion coefficient and the partial specific volume were measured and from these data the frictional ratio and the Stokes radius were calculated . These parameters indicate that the relatively high apparent molecular weight calculated from the gel-filtration experiments is caused by the assymetric shape of the inhibitor molecule rather than by an aggregation of subunits.

Eur J Biochem, 1978 Aug 1, 88(2), 425 - 31
Sulfite binding to a flavodehydrogenase, cytochrome b2 from baker's yeast; Lederer F; Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor . Yet it forms a red semiquinone {Capeillere Blandin et al . Eur . J . Biochem . 54, 549--566 (1975)} and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 muM) . This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance . A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum . It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite . It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavo-proteins may not be as general as previously proposed {Massey et al . J . Biol . Chem . 244, 3999--4006 (1969)}.

Biochim Biophys Acta, 1978 Jul 25, 530(1), 78 - 90
Triacylglycerol synthesis in lipid particles from baker's yeast (Saccharomyces cerevisiae); Christiansen K; Triacylglycerol synthesis has been studied in a lipid particle preparation of baker's yeast (Saccharomyces cerevisiae), and compared with the synthesis in other subcellular fractions . Fatty acid-CoA ligase (AMP) (EC 6.2.1.3) activity and sn-glycerol 3-phosphate acyltransferase activity (EC 2.3.1.15) were present in all the subcellular fractions tested but the highest specific activities of both enzymes were observed with the lipid particle fraction . The products of the glycerol 3-phosphate acylation indicate that triacyglycerol synthesis proceeds through the phosphatidic acid pathway . However, only a small and nearly constant amount of lysophosphatidic acid was found with the lipid particle fraction while the other subcellular fraction produced lysophosphatidic and phosphatidic acid with a more pronounced precursor/product relationship . Triacylglycerol synthesis from endogenous diacylglycerol present in the lipid particle was also demonstrated.

Eur J Biochem, 1978 Jun 1, 87(1), 85 - 99
Small-angle X-ray scattering on malate synthase from baker's yeast . The native substrate-free enzyme and enzyme-substrate complexes; Zipper P et al.; Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique . Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined . As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage . These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system . 1 . For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm . Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder . The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm . Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape . 2 . Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme . These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves) . Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness . These changes imply an increase of the axial ratio by 2.2--6.9%, i.e . substrate binding induces a decrease of anisometry . While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter . In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA.

Eur J Biochem, 1978 Jun 1, 87(1), 171 - 9
The synthesis of cytochrome b on mitochondrial ribosomes in baker's yeast; Lin LF et al.; Antiserum against a major cytochrome b peptide isolated from yeast mitochondria as described previously (Lin, L.-F.H., and Beattie, D.S., J . Biol . Chem . 1978, 253, 2412--2418) was raised in rabbits and shown to be monospecific against the pure antigen . Mitochondria were isolated from yeast cells grown in {3H}leucine, extracted with Lubrol and treated with antiserum to cytochrome b . Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of a single major band of molecular weight 31 000 corresponding to cytochrome b . In order to determine the intracellular site of translation of cytochrome b, yeast cells were labeled in vivo under non-growing conditions with {3H}leucine in the absence or presence of inhibitors of cytoplasmic and mitochondrial protein synthesis . The incorporation of radioactive leucine into the apoprotein of cytochrome b isolated by immunoprecipitation followed by gel electrophoresis was insensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and sensitive to acriflavin, erythromycin, and chloramphenicol (inhibitors of mitochondrial protein synthesis) . Furthermore, no cytochrome b apoprotein was present in a cytoplasmic petite mutant which lacked mitochondrial protein synthesis . Cytochrome b is thus a product of protein synthesis on mitochondrial ribosomes.

J Biol Chem, 1978 May 25, 253(10), 3666 - 70
Purification and characterization of saccharopine dehydrogenase from baker's yeast; Ogawa H et al.; Saccharopine dehydrogenase (N6-(glutar-2-yl)-L-ly-sine:NAD oxidoreductase (L-lysine-forming)) from baker's yeast was purified to homogenicity . The overall purification was about 1,200-fold over the crude extract with a yield of about 24% . The purified enzyme had a sedimentation coefficient (S20,w) of 3.0 S . The molecular weight determinations by sedimentation equilibrium, Sephadex G-100 gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a value of about 39,000 and, therefore, saccharopine dehydrogenase is a single polypeptide chain enzyme . A Stokes radius of 27 A and a diffusion constant of 7.9 X 10(-7) cm2 s-1 were obtained from Sephadex gel filtration chromatography . The enzyme had a high isoelectric pH of 10.1 . The NH2-terminal sequence was Ala-Ala---- . The enzyme possessed 3 cysteine residues/molecule; no disulfide bond was present . Incubation of saccharopine dehydrogenase with p-chloromercuribenzoate or iodoacetate resulted in complete loss of enzyme activity . Whereas the coenzyme and substrates were ineffective in protecting from inactivation by p-chloromercuribenzoate, iodoacetate inhibition was protected by excess coenzyme.

Eur J Biochem, 1978 May 16, 86(2), 399 - 406
Cytochrome c from Schizosaccharomyces pombe . 1 . Purification, spectral properties, and amino-acid composition; Claisse ML et al.; Cytochrome c from the fission yeast Schizosaccharomyces pombe has been purified . Its chromatographic and spectral properties are reported and compared to those of iso-1-cytochrome c from baker's yeast; the amino-acid composition is described . Schiz . pombe cytochrome c has a much lower affinity for Amberlite IRP64 than Sacch . cerevisiae iso-1-cytochrome c . Its alpha absorption band splits into three maxima (calpha1, calpha2, and calpha3) at -190 degrees C; this is unusual in yeasts, as shown by the low-temperature whole-cell absorption spectra which were examined in various yeast genera, species, and strains . A minor component can be separated by Amberlite chromatography . It exhibits the same low-temperature splitting of the alpha absorption band as the main fraction and it has a similar amino-acid composition with a notable exception: it is an unmethylated form of the cytochrome.

J Biol Chem, 1978 Apr 10, 253(7), 2412 - 8
Purification and properties of a major cytochrome b peptide from baker's yeast; Lin LF et al.; A major cytochrome b peptide was purified from yeast mitochondria by a procedure involving solubilization in deoxycholic and cholic acids, ammonium sulfate fractionation, proteolytic digestion, and sucrose gradient centrifugation in the presence of Tween 80 . The homogeneity of the purified protein was established by the criteria that the product was spectrally pure and yielded a single band on both sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by gel isoelectric focusing . The purified cytochrome b polypeptide had absorption maxima at 562, 532, and 430 nm in the reduced form and at 525 to 570 nm and 419 nm in the oxidized form . The reduced minus oxidized difference spectra revealed absorption bands at 562, 532, and 430 nm at room temperature and 559, 529, and 429 nm at 77 K, respectively . The heme group was identified as protoheme by formation of the reduced pyridine hemochromogen . Treatment of the reduced form with carbon monoxide affected the absorption spectrum, indicating that the isolated hemoprotein was modified compared to native cytochrome b . The apparent molecular weight of the preparation was 28,000 based on sodium dodecyl sulfate polyacrylamide-gel electrophoresis and 28,800 based on sucrose gradient centrifugation . The isolated cytochrome b polypeptide showed a strong tendency to aggregate.

J Biol Chem, 1978 Apr 10, 253(7), 2392 - 9
The preparation and characterization of highly purified, enzymically active complex III from baker's yeast; Siedow JN et al.; A soluble enzymically active cytochrome b.c1 complex has been purified from baker's yeast mitochondria by a procedure involving solubilization in cholate, differential fractionation with ammonium sulfate, and ultracentrifugation . The resulting particle is free of both cytochrome c oxidase and succinate dehydrogenase activities . The complex contains cytochromes b and c1 in a ratio of 2:1 and quinone and iron-sulfur protein in amounts roughly stoichiometric with cytochrome c1 . EPR spectroscopy has shown the iron-sulfur protein to be present mainly as the Rieske protein . EPR spectroscopy also shows a heterogeneity in the cytochrome b population with resonances appearing at g = 3.60 (cytochrome bK) and g = 3.76 (cytochrome bT) . A third EPR resonance appearing in the region associated with low spin ferric hemes (g = 3.49) is assigned to cytochrome c1 . Anaerobic titration of the complex with dithionite confirmed the heterogeneity in the cytochrome b population and demonstrated that the oxidation-reduction potential of the iron-sulfur protein is approximately 30 mV more positive than cytochrome c1 . An intense EPR signal assigned to the coenzyme Q free radical appeared midway in the reductive titration; this signal disappeared toward the end of the titration . A conformational change in the iron-sulfur protein attendant on reduction of a low potential species was noted.

Eur J Biochem, 1978 Mar 15, 84(2), 499 - 502
Threonyl-tRNA, lysyl-tRNA and arginyl-tRNA synthetases from Baker's yeast . Substrate specificity with regard to ATP analogues; Freist W et al.; Sixteen analogues of ATP have been tested in the aminoacylation reaction of threonyl-tRNA, lysyl-tRNA, and arginyl-tRNA synthetases from baker's yeast . Two compounds are substrates for threonyl-tRNA and for lysyl-tRNA synthetases and five compounds for arginyl-tRNA synthetase . There are six inhibitors for threonyl-tRNA, nine for lysyl-tRNA, and six for arginyl-tRNA synthetase . Their Km and Ki values have been determined . Thus positions 2, 6, 7, 8 and 9 of the purine moiety and 2' and 3' of the sugar moiety of the ATP molecule are important for catalytic action of these aminoacyl-tRNA synthetases . Remarkably arginyl-tRNA synthetase is the first aminoacyl-tRNA synthetase which tolerates bulky substituents at the sugar moiety of ATP . These data fit with the idea that synthetases of subunit structure need magnesium-ion-ATP complexes with an anti conformation as substrates whereas single-chain enzymes accept this substrate in the syn conformation.

Biochem J, 1978 Mar 1, 169(3), 505 - 8
The presence of dolichol in a lipid diphosphate N-acetylglucosamine from Saccharomyces cerevisiae (baker's yeast); Reuvers F et al.; The lipid moiety of a lipid diphosphate N-acetylglucosamine, an intermediate in glycosylation of proteins, was studied . Ozonolysis of the compound gave evidence for an alpha-saturated isoprene unit . Alkaline hydrolysis of the glycolipid, followed by high-pressure liquid chromatography, showed the presence of a series of polyprenol homologues identical with those isolated directly from Saccharomyces cerevisiae (baker's yeast) . No particular homologue was preferred in the enzymic transfer of N-acetylglucosamine 1-phosphate to endogenous dolichol monophosphate.

Biokhimiia, 1978 Mar, 43(3), 545 - 54
{Molecular weight and tertiary structure of transketolase from baker's yeast}; Beliaeva RKh et al.; Molecular weight of native apotransketolase from baker's yeast is found to be 159000 +/- 6000 by means of sedimentation equilibrium and sedimentation-diffusion rate . The enzyme in a relatively low concentration reversibly dissociates into two subunits with molecular weight of about 80 000 at pH 7.6 and 20 degrees C . The equilibrium constant of the reaction monomer-dimer is 4.4 . 10(3) M-1 . A decrease of the temperature stimulates the association of monomers into dimer, while the shift of pH 7.6 into acid or alkaline region stimulates the dissociation process . Dissociation becames irreversible at pH less than 5 and greater than 10.5.

J Biochem (Tokyo), 1978 Feb, 83(2), 585 - 97
Successive purification of several enzymes having affinities for phosphoric groups of substrates by affinity chromatography on P-cellulose; Takagahara I et al.; Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and pyruvate kinase of Candida utilis and baker's yeast, when in anionic form, were adsorbed on a cation exchanger, P-cellulose, due to affinities similar to those for the phosphoric groups of their respective substrates; thus, glucose-6-phosphate dehydrogenase was readily eluted by either NADP+ or NADPH, glutathione reductase by NADPH, 6-phosphogluconate dehydrogenase by 6-phosphogluconate, and pyruvate kinase by either ATP or ADP . This type of chromatography may be called "affinity-adsorption-elution chromatography"; the main principle is different from that of so-called affinity-elution chromatography . Based on these findings, a large-scale procedure suitable for successive purification of several enzymes having affinities for the phosphoric groups of their substrates was devised . As an example, glucose-6-phosphate dehydrogenase was highly purified from baker's yeast and crystallized.

Zentralbl Bakteriol Naturwiss, 1978, 133(7-8), 726 - 32
The effect of the water activity of the milieu on rates of glucose uptake by the osmophilic yeasts Saccharomyces rouxii and Debaryomyces hansenii; Mahmoud MI; Rates of glucose uptake in baker's yeast and in the osmophilic yeasts D . hansenii and S . rouxii were investigated at different values of water activity of the milieu, as regulated either by glycerol or sodium chloride . In both cases, D . hansenii could maintain relatively higher rates of glucose uptake . At lower values of water activity, sodium chloride exerted an inhibitory effect on rates of glucose uptake by S . rouxii, while in the presence of glycerol, rates of glucose uptake shown by S . rouxii resembled those shown by D . hansenii . Rates of glucose uptake by baker's yeast were drastically affected at lower values of water activity in the presence of either solute . Lower values of water activity exerted a stimulatory effect on catalase activity of both S . rouxii and D . hansenii . However, activities of baker's yeast with regard to catalase and invertase were moderately affected under such conditions . Results presented may lead to the presumption that osmophilic yeasts, at least partly, have solved the problem of osmotic tolerance over nonosmotolerant strains by possessing a high capacity for maintaining higher rates of glucose uptake, in spite of the adverse external concentration of solute.

Prikl Biokhim Mikrobiol, 1978 Jan-Feb, 14(1), 60 - 6
{Amino acid makeup of preparations from baker's yeast autolysates}; Belikov VM et al.; Yeast autolysis and hydrolysate composition were studied with respect to the autolysis of baker's pressed yeast . A method of isolating a mixture of amino acids and lower peptides from autolysates was developed, using ion-exchange resins . The above compounds reached maximum accumulation by the 15-20th hour of autolysis . A mixture of amino acids applicable to nutrition, including dietotherapy, was prepared.

Acta Biol Med Ger, 1978, 37(3), 375 - 85
Fluorimetric characterization and influence of Cu2+ binding on the fluorescence of inorganic pyrophosphatase from baker's yeast; Hohne WE et al.; The denaturation characteristics of inorganic pyrophosphatase from baker's yeast and the interaction with Cu2+ were investigated with fluorimetric methods . The position of the fluorescence emission spectrum with a maximum at 328 nm together with a quantum yield of 0.12 led to the conclusion that most of the tryptophan residues of the protein are buried in nonpolar inner regions of the molecule . The contribution of the tyrosine residues to the fluorescence of pyrophosphatase is only about 7% . Denaturation of the protein with denaturants or changes of the pH value cause a red shift of the fluorescence emission maximum . In the presence of Cu2+ ions a fluorescence quenching is observed . Thereby, a specific binding of one Cu2+ per subunit may be distinguished from further unspecific Cu2+ binding . The Cu2+ binding to the latter sites shows a time dependence according to a slow, reversible exposure of additional binding sites . This time dependent binding characteristics was also verified by following the free Cu2+ concentration with the fluorescent "metal indicator" epsilon-ADP.

Acta Chem Scand B, 1978, 32(6), 447 - 51
On the properties of alpha-glucosidase and the binding of glucose to the enzyme; Siro MR et al.; An alpha-glucosidase was purified from baker's yeast . The molecular weight was approximately 44 000 daltons . SDS-disc gel electrophoresis suggested that the enzyme consisted of four subunits . The isoelectric point was at pH 5.4 . The Km values for p-nitrophenyl alpha-D-glucopyranoside and maltose were 2.9 X 10(-4) and 2.5 X 10(-2) M, respectively . Binding of 2-(p-toluidino)naphthalene-6-sulfonate to the alpha-glucosidase was associated with a strong increase in fluorescence . The dissociation constant of the enzyme-TNS complex was 8 X 10(-5) M . The fluorescent probe did not interfere with the binding of glucose to the enzyme although the alpha-glucosidase was inhibited by high concentrations of TNS . The formation of an enzyme-glucose complex was indicated by an increase of fluorescence and by a shift in the wavelength for maximal emission which suggests that the binding process is associated with a change in conformation . The dissociation constant of the glucose--alpha-glucosidase complex KD = 0.57 X 10(-3) M, was calculated from the increase in fluorescence as a function of glucose concentration.

Acta Biol Med Ger, 1978, 37(1), 13 - 7
The interaction of uranyl ions with inorganic pyrophosphatase from baker's yeast; Bienwald B et al.; The interaction of uranyl ions with inorganic pyrophosphatase from baker's yeast was investigated by measurement of their effect on the protein fluorescence . Fluorescence titrations of the native enzyme with uranyl nitrate show that there is a specific binding of uranyl ions to the enzyme . It was deduced that each subunit of the enzyme binds one uranyl ion . The binding constant was estimated to be in the order of 10(7) M-1 . The enzyme which contains a small number of chemically modified carboxyl groups was not able to bind uranyl ions specifically . The modification of carboxyl groups was carried out by use of a water soluble carbodiimide and the nucleophilic reagent N-(2,4-dinitro-phenyl)-hexamethylenediamine . The substrate analogue calcium pyrophosphate displaced the uranyl ions from their binding sites at the enzyme . From the results it is concluded that carboxyl groups of the active site are the ligands for the binding of uranyl ions.

Folia Microbiol (Praha), 1978, 23(2), 118 - 25
Apparent half-lives of sugar transport proteins in Saccharomyces cerevisiae; Alonso A et al.; Using incubation in the presence of 0.4 mM cycloheximide the half-lives of the principal membrane transport proteins in baker's yeast were found to be: more than 24 h for the constitutive glucose carrier, 2.2 h for the inducible galactose carrier, 1.2 h for the inducible maltose carrier and 0.8 h for the inducible alpha-methyl-D-glucoside carrier . The distinct nature of the two last-named carriers was thus supported . De-induction of the galactose carrier was enhanced in the presence of glucose plus cycloheximide but not of either substance alone . Chloramphenicol suppressed all effects of cycloheximide . In contrast to the enzymes of galactose metabolism, the induction of the glactose carrier was not under the control of a mitochondrial factor and took place in a rho-mutant . The system induced by maltose but not the one induced by alpha-methyl-D-glucoside was de-induced rapidly by the intervention of a cytoplasm-synthesized protein.

Acta Biol Med Ger, 1978, 37(7), 1129 - 33
Kinetic studies of the interaction of uranyl ions with inorganic pyrophosphatase from baker's yeast; Bienwald B et al.; Kinetic measurements were performed to test the effect of uranyl ions on the enzymatic hydrolysis of pyrophosphate . A strong inhibition of the enzyme was found . From a Dixon-plot an inhibition competitive to the substrate magnesium pyrophosphate and an inhibitory constant of Ki = 3 . 10(-7) M was deduced.

Acta Biol Med Ger, 1978, 37(4), 513 - 7
{Small-angle scattering of the quaternary structure of phosphofructokinase from baker's yeast}; Plietz P et al.; The phosphofructokinase (E.C . 2.7.1.11) from baker's yeast was examined by means of small angle X-ray scattering in 0.1 M K-phosphate buffer, pH 7 . A quaternary structure model was obtained from the comparison of the model scattering curve with the experimental one . The eight subunits of the yeast phosphofructokinase are arranged in a D2-symmetry . The proposed scattering equivalent model corresponds to a structural description with a resolution of 2.5 nm . Models with a C8-symmetry and D4-symmetry can be ruled out.

Folia Microbiol (Praha), 1978, 23(6), 409 - 22
Effect of inhibitors on acid production by baker's yeast; Sigler K et al.; Glucose-induced acid extrusion, respiration and anaerobic fermentation in baker's yeast was studied with the aid of sixteen inhibitors . Uranyl(2+) nitrate affected the acid extrusion more anaerobically than aerobically; the complexing of Mg2+ and Ca2+ by EDTA at the membrane had no effect . Inhibitors of glycolysis (iodoacetamide, N-ethylmaleimide, fluoride) suppressed acid production markedly, and so did the phosphorylation-blocking arsenate . Fluoroacetate, inhibiting the citric-acid cycle, had no effect . Inhibition by uncouplers depended on their pKa values: 2,4,6-trinitrophenol (pKa 0.4) less than 2,4-dinitrophenol (4.1) less than azide (4.7) less than 3-chlorophenylhydrazonomalononitrile (6.0) . Inhibition by trinitrophenol was only slightly increased by its acetylation . Cyanide and nonpermeant oligomycin showed practically no effect; inhibition by dicyclohexylcarbodiimide was delayed but potent . The concentration profiles of inhibition of acid production differed from those of respiration and fermentation . Thus, though the acid production is a metabolically dependent process, it does not reflect the intensity of metabolism, except partly in the first half of glycolysis.

Biokhimiia, 1978 Jan, 43(1), 50 - 7
{Catalytic properties of three isoenzymes possessing pyrophosphatase activity, isolated from baker's yeast}; Kasho VN et al.; A kinetic study of inorganic pyrophosphatase isolated from brewer's yeast was done . It was shown that all three isoenzymes have the same pH-optimum and specificity with respect to substrate and metal activator . Statistical treatment of the kinetic data yielded equilibrium and catalytical constants, describing enzyme interaction with the metal activator and substrate . The catalytic properties of all three isoenzymes are similar to those of the baker's yeast pyrophosphatase . The fluoride inhibition pattern for inorganic pyrophosphatase from brewer's yeast is similar to that for the baker's yeast enzyme.

Mol Cell Biochem, 1977 Nov 25, 18(1), 21 - 7
Molecular properties of yeast glucokinase; Maitra PK et al.; Glucokinase from baker's yeast has been purified to homogeneity . The molecular weight of the subunit is 51,000 . The native enzyme sediments with S20,w values in the range of 19 to nearly 4S . The presence of glucose and phosphate favors the heavier species while ATP causes depolymerization . Titration experiments with the Ellman reagent support this view . The enzyme subunit has four sulfhydryl residues of which one is more reactive than the other three . However, it does not seem to be directly responsible for the catalytic activity . The amino acid composition of the enzyme is similar to those of the hexokinases P1 and P2 but for aspartic acid and histidine.

J Biochem (Tokyo), 1977 Nov, 82(5), 1325 - 9
Origin of hydrogen atoms in the fatty acids synthesized with yeast fatty acid synthetase; Seyama Y et al.; The mechanism of hydrogen incorporation into fatty acids was investigated with an enzyme preparation from baker's yeast . Fatty acids synthesized from malonyl-CoA and acetyl-CoA in the presence of D2O or stereospecifically deuterium-labeled NADPH were isolated and analyzed by mass chromatography to examine the localization of deuterium atoms in the molecule . The following results were obtained: 1 . Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) . The second hydrogen atom was incorporated as the result of hydrogen exchange phenomenon between the methylene group of malonyl CoA and water . 2 . HB hydrogen of NADPH was used for beta-ketoacyl reductase . 3 . HB hydrogen of NADPH was also used for enoyl reductase . 4 . Hydrogen atoms from HB position of NADPH were found on the odd-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom).

Ital J Biochem, 1977 Nov-Dec, 26(6), 473 - 86
Polymeric forms of yeast nicotinamide-adenine dinucleotide-specific isocitrate dehydrogenase in the presence of various ligands; Konig G et al.; The molecular weight of NAD-specific isocitrate dehydrogenase, purified from baker's yeast, has been studied by molecular sieve chromatography . By elution of the enzyme from columns of Sepharose 6B with 0.05 M phosphate buffer, pH 7.6, a mol . wt of 151,000 was measured . Higher values of the mol . wt were measured in presence of the following ligands (mol . wt in parentheses): the substrate, isocitrate (224,000); the activators, citrate (203,000) and AMP (275,000); the inhibitor, NaCl (360,000) . A mol . wt of 337,000 was measured when AMP, which antagonizes the inhibition by chloride, was present together with NaCl . The results indicate the absence of a correlation between the aggregation form of the enzyme in presence of the ligands and the effects of these ligands on the enzyme activity.

Biochim Biophys Acta, 1977 Oct 13, 484(2), 268 - 74
Purification by affinity chromatography of yeast glutathione reductase, the enzyme responsible for the NADPH-dependent reduction of the mixed disulfide of coenzyme A and glutathione; Carlberg I et al.; Glutathione reductase (NAD(P)H : oxidised-glutathione oxidoreductase, EC 1.6.4.2) was purified from baker's yeast by a new procedure involving affinity chromatography on 2',5'-ADP-Sepharose 4B . The yield was 65% of essentially homogeneous enzyme . The activity was assayed with both glutathione disulfide (GSSG) and the mixed disulfide of coenzyme A and glutathione (CoAssg) . The two disulfide substrates gave coinciding activity profiles and a constant ratio of the activities in different chromatographic and electrophoretic systems . No evidence was obtained for the existence of a reductase specific for CoASSG distinct from glutathione reductase . It is concluded that normal baker's yeast contains a single reductase active with both GSSG and CoASSG.

Ann Rheum Dis, 1977 Oct, 36(5), 433 - 41
Cellular immunity in systemic lupus erythematosus as evidenced in vitro by leucocyte migration inhibition tests; Okubo M et al.; A leucocyte migration inhibition test was performed on 26 patients with systemic lupus erythematosus (SLE) and on 35 control subjects using three different antigens, fetal calf thymus DNA, baker's yeast RNA, and calf thymus extractable nuclear antigen (ENA) . Leucocyte migration was inhibited by DNA in 17 out of 26 SLE patients (65-3%), and in only 2 of the 35 controls (5-7%) . When RNA or ENA was added none of the patients or controls showed inhibition . In SLE patients migration inhibition by DNA was significantly correlated with the presence of proteinuria and/or granular casts in urinary sediment . When the migration inhibition test was positive, immunofluorescence verified active histology of the glomeruli obtained by a percutaneous renal biopsy.

Nucleic Acids Res, 1977 Sep, 4(9), 3239 - 58
Primary structure of baker's yeast tRNAVal 2b; Gorbulev VG et al.; The minor form of valine tRNA from baker's yeast-tRNAVal 2b--purified by column chromatography was completely digested with guanylo-RNase and pancreatic RNase . The products of these digestions were separated by a combination of thin-layer chromatography on cellulose and high voltage electrophoresis on DEAE-paper and then identified . The halves of tRNA Val 2b were prepared by partial digestion with pancreatic RNase, and their complete guanylo-RNase and pancreatic RNase digests were analysed . Basing on the obtained data the primary structure of baker's yeast tRNA Val 2b was reconstructed.

Carbohydr Res, 1977 Aug, 57, 3 - 13
Purification of some glycoside hydrolases by affinity chromatography; Edward M et al.; Two glycoproteins have been isolated from the cell walls of baker's yeast . One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1 leads to 3)-linked beta-D-glucosyl residues, some of which are attached by (1 leads to 6)-linkages to the main chain . Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1 leads to 3)-beta-D-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806 . The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an alpha-D-mannosidase from jack-bean meal.

Biochemistry, 1977 Jul 26, 16(15), 3429 - 32
Stereochemistry of internucleotidic bond formation by tRNA nucleotidyltransferase from baker's yeast; Eckstein F et al.; Isomer A of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) is a substrate for tRNA nucleotidyltransferase from baker's yeast, whereas isomer B is a competitive inhibitor . The tRNA resulting from this reaction has a phosphorothioate instead of a phosphate diester linkage at the last internucleotidic linkage between cytidine and adenosine . On limited digestion of this tRNA with RNase A, one can isolate cytidine 2',3'-cyclic phosphorothioate which can be deaminated to uridine 2',3'-cyclic phosphorothioate . It can be shown that this compound is the endo isomer and that, therefore, the phosphorothioate diester bond in the tRNA must have had the R configuration . This result indicates that no racemization during the condensation of ATP alpha S, isomer A, onto the tRNA had occurred . Whether inversion or retention of configuration had taken place awaits elucidation of the absolute configuration of isomer A of ATP alpha S.

Biochem J, 1977 Jul 1, 165(1), 149 - 55
Purification and steady-state kinetics of adenosine 5'-pyrophosphate sulphurylase from baker's yeast; Nicholls RG; ADP sulphurylase (EC 2.7.7.5) was purified by chromatography on Sephadex G-200 and DEAE-cellulose . The enzyme was assayed by measuring the incorporation of {32P}Pi into ADP in the presence of the substrate for the reverse reaction, adenosine 5'-sulphatophosphate . In the concentration ranges investigated, by using initial-velocity, product-inhibition and isotope-exchange studies, the data were consistent with a Ping Pong reaction mechanism, with Km for adenosine 5'-sulphatophosphate of 1.20 +/- 0.08 mM and a Km for Pi of 4.95 +/- 0.15 mM . Competitive substrate inhibition by Pi (Ki = 11.7 +/- 0.3 mM) was found . ADP sulphurylase catalyses a sulphate-independent Pi-ADP exchange reaction, the kinetics of which are consistent with the kinetics of the overall reaction, inconsistent with the assay of Burnell & Anderson {(1973) Biochem . J . 133, 417-428}, which is based on a sulphate-dependent Pi-ADP exchange reaction.

Science, 1977 Jun 3, 196(4294), 1115 - 7
Serum complement-like opsonic activities in human, animal, vegetable, and proprietary milks; Miller ME et al.; Human, animal, proprietary, and soy milks are comparable to human serum C5 in opsonization of baker's yeast . Bovine milk and human serum opsonically reconstitute C5-deficient mouse serum . Such reconstitution is selectively inhibited by antiserum to human C5 . Further characterization suggests that bovine milk contains material structurally and functionally similar, but not identical, to human C5.

Eur J Biochem, 1977 Jun 1, 76(1), 263 - 7
On the stereochemistry of activation of phenylalanine by phenylalanyl-tRNA synthetase from baker's yeast; von der Haar F et al.; Because of its chiralic alpha-phosphorus atom adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS) exists in two diastereomeric forms, arbitrarily named (A) and (B) . For phenylalanyl-tRNA synthetase ATPalphaS (A) is a substrate whereas ATPalphaS (B) is neither a substrate nor an inhibitor . During the ATPalphaS (A)/PPi exchange reaction with phenylalanyl-tRNA synthetase the configuration at the alpha-phosphorus is retained . The mechanistic implications of these findings are discussed . Preliminary investigations with several other aminoacyl-tRNA synthetases show that the stereochemical requirement with respect to the alpha-phosphorus of ATP is not identical for all aminoacyl-tRNA synthetases.

Biochem J, 1977 Jun 1, 163(3), 409 - 10
The separation of oligonucleotides of baker's-yeast value transfer ribonucleic acid 2b by high-voltage electrophoresis on DEAE-paper and by thin-layer chromatography; Gorbulev VG et al.; A modified procedure for the separation of oligoribonucleotides is described that is based on the combination of t.l.c . on cellulose and electrophoresis on DEAE-paper at 4000 V on a cooling plate . The technique is relatively rapid and allows the analysis of larger quantities than is possible by electrophoresis on cellulose acetate.

Biochem J, 1977 Jun 1, 163(3), 467 - 76
Characterization of an adenosine 3':5'-cyclic monophosphate phosphodiesterase from baker's yeast . Its binding to subcellular particles, catalytic properties and gel-filtration behaviour; Londesborough J; 1 . The 3':5'-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP . Catalytic activity is abolished by 30 micrometer-EDTA . At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer . The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase {PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem . Scand . 26, 3396-3398}.2 . At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl . MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions . In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3 . At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C . These observations suggest that the binding is electrostatic rather than hydrophobic . 3 . The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26 . Presumably a substantial amount of the enzyme is particle-bound in vivo . 4 . At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000 . In the presence of 0.5M-KCl, a constant mol.wt . of about 55 000 was observed over a 20-fold range of enzyme concentrations.

Biochemistry, 1977 May 17, 16(10), 2202 - 7
Enzymic formation of glycolate in Chromatium . Role of superoxide radical in a transketolase-type mechanism; Asami S et al.; Chromatophores prepared from Chromatium exhibit a light-dependent O2 uptake in the presence of reduced 2,6-dichlorophenolindophenol, the maximum rate observed being 10.8 micronmol (mg of Bchl)-1 h-1 (air-saturated condition) . As it was found that the uptake of O2 was markedly inhibited by superoxide dismutase, it is suggested that molecular oxygen is subject to light-dependent monovalent reduction, resulting in the formation of the superoxide anion radical (O2-) . By coupling baker's yeast transketolase with illuminated chromatophore preparations, it was demonstrated that {U-14C}-fructose 6-phosphate (6-P) is oxidatively split to produce glycolate, and that the reaction was markedly inhibited by superoxide dismutase and less strongly by catalase . A coupled system containing yeast transketolase and xanthine plus xanthine oxidase showed a similar oxidative formation of glycolate from {U-14C} fructose 6-P . It is thus suggested that photogenerated O2- serves as an oxidant in the transketolase-catalyzed formation of glycolate from the alpha, beta-dihydroxyethyl (C2) thiamine pyrophosphate complex, whereas H2O2 is not an efficient oxidant . The rate of glycolate formation in vitro utilizing O2- does not account for the in vivo rate of glycolate photosynthesis in Chromatium cells exposed to an O2 atmosphere (10 micronmol (mg of Bchl)-1 h-1) . However, the enhancement of glycolate formation by the autoxidizable electron acceptor methyl viologen in Chromatium cells in O2, as well as the strong suppression by 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an O2- scavenger, suggest that O2- is involved in the light-dependent formation of glycolate in vivo.

Eur J Biochem, 1977 May 16, 75(2), 561 - 70
Tyroslyl-tRNA synthetase from baker's yeast . Rapid isolation by affinity elution, molecular weight of the enzyme, and determination of essential sulfhydryl groups; Faulhammer HG et al.; Tyrosyl-tRNA synthetase (EC 6.1.1.1) has been isolated from baker's yeast with an overall purification factor of more than 5000 . After opening the cells, pH 4.8 precipitation, ammonium sulfate fractionation, removal of the nucleic acids with DEAE-cellulose and chromatography on CM-Sephadex, the critical purification step is the elution of the cation-exchanger-bound tyrosyl-tRNA synthetase with tRNATyr . The homogeneous enzyme exhibits a molecular weight of 40 000 as estimated by sedimentation equilibrium centrifugation and dodecylsulfate-gel electrophoresis under reducing and non-reducing conditions . Gel filtration experiments show a molecular weight of about 100 000 indicating the existence of an active dimeric form . The possibility of proteolytic cleavage of the enzyme is excluded . The reaction of tyrosyl-tRNA synthetase with p-chloromercuribenzoate and N-ethylmaleimide reveals two repidly reacting sulfhydryl groups per subunit of molecular weight 40 000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme-inhibitor complexes . In addition an efficient purification method is described for isolating tRNATyr from soluble ribonucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.

Arch Microbiol, 1977 May 13, 113(1-2), 159 - 61
Location of three key enzymes of gluconeogenesis in baker's yeast; Haarasilta S et al.; The subcellular location of hexose diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in baker's yeast (Saccharomyces cerevisiae) was investigated by density gradient centrifugation of spheroplast lysates obtained by osmotic shock treatment of spheroplasts and centrifugation for 10000 g x min . On the evidence obtained from zonal separations these three enzymes of gluconeogenesis are most probably located in the soluble cytosol.

Proc Natl Acad Sci U S A, 1977 May, 74(5), 1988 - 92
Dynamic structure of whole cells probed by nuclear Overhauser enhanced nitrogen-15 nuclear magnetic resonance spectroscopy; Lapidot A et al.; The proton-decoupled 15N Fourier transform nuclear magnetic resonance (NMR) spectra of 15N-enriched Escherichia coli, Bacillus licheniformis, baker's yeast, and Friend leukemic cells were obtained . The 15N NMR spectra of whole cells displayed 15N resonances originating from (i) protein backbones with lysine, arginine, and histidine side chains, (ii) ribonucleic acids, (iii) peptidoglycan, and (iv) phospholipids . Several additional amino and amide resonances were observed but not identified . In bacteria and yeast, the cell wall was found to be the site of a relatively mobile group of molecules, whose resonances dominate the proton-decoupled 15N NMR spectra of whole cells . 15N NMR chemical shifts and nuclear Overhauser effects have provided information on the in vivo structure of cell wall peptidoglycan . In Staphylococcus aureus the pentaglycine cross-bridge of cell wall peptidoglycan was found to have a random coil conformation . In B . licheniformis considerable segmental motional freedom was detected in teichuronic acid and peptidoglycan polysaccharide chains in the wall of the intact cell.

Biochemistry, 1977 Apr 19, 16(8), 1696 - 702
Hydrolytic action of aminoacyl-tRNA synthetases from baker's yeast . "Chemical proofreading" of Thr-tRNA Val by valyl-tRNA synthetase studied with modified tRNA Val and amino acid analogues; Igloi GL et al.; The properties of native and of two modified tRNA Val species in the correction of misactivated threonine by valyl-tRNA synthetase have been studied . Whereas Thr-tRNA Val-C-C-A could not be isolated in the valyl-tRNA synthetase catalyzed reaction, Thr-tRNA Val-C-C-3'dA is isolable in up to 50% yield in this system and tRNA Val-C-C-3'NH2A is fully aminoacylated with threonine by the same enzyme . The hydrolysis of preformed Thr-tRNA Val-C-C-A by free valyl-tRNA synthetase is 30 times faster than the corresponding breakdown of Val-tRNA Val-C-C-A . This hydrolytic activity is also observed with Thr-tRNA Val-C-C-3'dA although the rate is reduce to that of the reaction of Val-tRNA Val-C-C-A . Modification of the threonine to O-methylthreonine, which is also a substrate for valyl-tRNA synthetase, leads to stabilization of the O-methylthreonyl-tRNA esters . The AMP/PP independent hydrolysis under aminoacylating conditions, which is a measure of the correction process, indicates that O-MeThr-tRNA Val-C-C-A is only very slowly corrected while the tRNA Val-C-C-3'dA and tRNA Val-C-C-3'NH2A esters are completely stable . Removal of the methoxy group of O-methylthreonine as in alpha-amino-butyric acid increases the rate of the hydrolytic reaction and once again alpha-Abu-tRNA Val-C-C-A and alpha-Abu-tRNA Val-C-C-3'dA are unstable under aminoacylating conditions and not isolable.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1343 - 7
Chromatin subunits from baker's yeast: isolation and partial characterization; Nelson DA et al.; The organization of proteins along DNA in chromatin of Saccharomyces cerevisiae (baker's yeast) was examined by analyzing the DNA and nucleoprotein products obtained after digestion of yeast nuclei with staphylococcal nuclease . Yeast DNA is digested in situ at regularly spaced cleavage sites about 160 base pairs apart . Nucleoprotein fragments were resolved and isolated by centrifugation on linear, 5-20% sucrose gradients . The predominant 11S component appears to be identical to chromatin "subunits" or "nucleosomes" isolated from higher eukaryotes, containing a 150-160 base pair length of DNA and approximately equimolar amounts of four proteins that coelectrophorese with calf histones H2A, H2B, H3, and H4, plus small amounts of three proteins that electrophorese similarly to H1 histones . Thus, the structural organization of the yeast genome is similar to that of more developed organisms, except for the smaller total repeat length . None of the yeast subunit proteins, including the possible H1 proteins, contains cysteine.

Eur J Biochem, 1977 Mar 1, 73(2), 443 - 7
Bromopyruvate as an affinity label for baker's yeast flavocytochrome b2 . Kinetic study of the inactivation reaction; Mulet C et al.; Bromopyruvate was shown to completely inactivate cytochrome b2 in a reaction that obeyed the kinetic criteria required for affinity labels: it inactivated flavocytochrome b2 according to saturation kinetics, and the inactivation reaction was competitively inhibited by the substrate or competitive inhibitors . Inactivation was irreversible . The behaviour of both forms of flavocytochrome b2 (lintact and proteolytically cleaved) was examined . It was found that the reduced cleaved enzyme was not inactivated by bromopyruvate; this phenomenon can probably be ascribed to a structural change undergone upon reduction . The value of the lactate dissociation constant of intact cytochrome b2 cytochrome b2 was determined in competition experiments with bromopyruvate . By comparison with the divergent published values for the Ks of the cleaved from, it appears that only those that differ from the Km by a factor of two or three are reasonable . This study opens the way for the identification of an active site residue and localization in the peptide chain of the bifunctional enzyme.

Biochim Biophys Acta, 1977 Feb 16, 474(4), 619 - 28
A factor affecting stimulation of aminocylation in plants; Bartkowiak S et al.; The presence of a factor stimulating the reaction of aminoacylation tRNAs was found in the seeds of lupin, with a molecular weight of 950 as estimated by gel filtration . The influence of this factor on the kinetics of the aminoacylation reactions of lupin, Escherichia coli, Baker's yeast and heterogeneous systems was investigated . This factor inhibits the esterification reaction of aminoacyl-tRNA synthetases from bacteria and yeast . Its influence on the optimum pH activity of isoleucyl-tRNA synthetase from lupin was determined.

Mol Cell Biochem, 1977 Feb 4, 14(1-3), 81 - 6
Structure of cytochrome c oxidase from baker's yeast - a progress report . Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochrome c binding site; Birchmeier W; Cytochrome c oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K) . In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtration . Their amino acid composition as well as their amino- and carbosy-terminal amino acid residues have been determined . Sequence determinations of subunits IV and VI are already in an advanced state . The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids . The binding site of yeast cytochrome oxidase for cytochrome c was studied by chemical crosslinking experiments . The formation of a disulfide bridge between the two proteins was observed by using cytochrome c from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107 . Alternatively, a disulfide between yeast cytochrome c and the oxidase could be formed directly by oxidation with copper phenanthroline . Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K . This new band appears to be derived from cytochrome c and from subunit III of cytochrome oxidase.

Nucleic Acids Res, 1977, 4(5), 1633 - 47
1H NMR studies of transfer RNA III: the observed and the computed spectra of the hydrogen-bonded NH resonances of baker's yeast transfer-RNA Phe; Kan LS et al.; The hydrogen-bonded NH resonances of Baker's yeast tRNAphe in H2O solution with Mg++ have been measured by a 360 MHz spectrometer at 23 degrees C . Totally, fifteen peaks and one shoulder can be resolved which represent 25 +/- 1 protons . Based on the refined atomic coordinates of the tRNAphe in the orthorhombic crystal, on the recent advances in the distance dependence of the ring-current magnetic field effects and on the adopted values for the isolated hydrogen-bonded NH resonances, a computed spectrum consisting of 23 protons was constructed . A quantitative comparison by computer was made between the computed spectrum and the spectrum simulated from the observed spectrum . These two spectra are closely similar but not identical . We suggest that the conformation of yeast tRNAphe in aqueous solution is closely similar but not identical to that found in the crystal, especially in the T psi C region and D region . Also the NH resonances in 3-4 proposed hydrogen bonds (most likely for tertiary structure) may exchange very rapidly in aqueous solution.

Blood, 1977 Jan, 49(1), 125 - 37
Applicability of an enzymatic quantitation of methylmalonic, propionic, and acetic acids in normal and megaloblastic states; Frenkel EP et al.; A rapid sensitive spectrophotometric assay for the measurement of methylmalonic and propionic acids in urine is described . The assay is based upon the quantitation of propionic acid using acetyl coenzyme A synthetase isolated from baker's yeast . This enzyme is highly specific for acetate and propionate, and acetate interference is eliminated by conversion to citrate . Methylmalonic acid was assayed by converting it to propionate by heat decarboxylation and then measuring the propionate increment over the endogenous amount in the noncarboxylated sample . Studies of urine obtained from normal subjects (by isolation, partial purification, and then assay by the isotope dilution technique) demonstrated urinary excretion of less than 1 mg of propionic acid and 1-5 mg of methylmalonic acid per day . In 22 consecutive patients with documented vitamin B12 deficiency, methylmalonic acid excretion in excess of 30 mg/24 hr was found . In four other patients, with only neurologic involvement methylmalonic aciduria aided in identifying B12 deficiency as an etiologic factor . Methylmalonic acid excretion was measured by direct assay of an aliquot of urine, requiring neither a valine load nor special extraction procedures . Propionic aciduria was variably increased in B12 deficiency and did not correlate either with the severity of the deficit or degree of methylmalonic aciduria . The assay was performed on urine, but it is potentially applicable to tissue extracts . In addition, this assay method can be utilized for the quantification of urine acetate levels as well.

Z Allg Mikrobiol, 1977, 17(6), 465 - 80
Enzymes of the yeast lytic system produced by Arthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls; Vrsanska M et al.; Yeast lytic system produced by Arthrobacter GJM-1 bacterium during growth on baker's yeast cell walls contains a complete set of enzymes which can hydrolyze all structural components of cell walls of Saccharomyces cerevisiae . Chromatographic fractionation of the lytic system showed the presence of two types of endo-beta-1,3-glucanase . Rapid lysis of isolated cell walls of yeast was induced only by endo-beta-1,3-glucanase exhibiting high affinity to insoluble beta-1,3-glucans and releasing laminaripentaose as the main product of hydrolysis of beta-1,3-glucans . This enzyme was able to lyse intact cells of S . cerevisiae only in the presence of an additional factor present in the Arthrobacter GJM-1 lytic system, which was identified as an alkaline protease . This enzyme possesses the lowest molecular weight among other identified enzyme components present in the lytic system . Its role in the solubilization of yeast cell walls from the outer surface by endo-beta-1,3-glucanase could be substituted by preincubation of cells with Pronase or by allowing the glucanase to act on cells in the presence of thiol reagents . The mechanism of lysis of intact cells and isolated cell walls by the enzymes of Arthrobacter GJM-1 is discussed in the light of the present conception of yeast cell wall structure.

Folia Microbiol (Praha), 1977, 22(5), 360 - 2
Specificity of trans-inhibition of amino acid transport in baker's yeast; Horak J et al.; The trans-inhibition potency of intracellular amino acids on the transport of various amino acids follows the same sequence, viz . Pro(Lys), Phe, Glu, Ala, Gly, Leu, and alpha-aminoisobutyric acid . The same sequence was found for the reciprocal of trans-inhibition constants . It appears that the intracellular amino acid itself or a derivative thereof acts on a component that is common to all amino acid transport systems of baker's yeast.

Biochimie, 1977, 59(5-6), 453 - 62
The yeast aminoacyl-tRNA synthetases . Methodology for their complete or partial purification and comparison of their relative activities under various extraction conditions; Kern D et al.; Several fractionation steps are described which can be applied to the partial purification of the 20 aminoacyl-tRNA synthetases from commercial baker's yeast . Comparative experiments performed in the presence or absence of protease inhibitors revealed that some enzymes prepared in the presence of the inhibitor exhibit much higher specific activities than the proteins extracted in the absence of the inhibitor . The methodology reported can be used for the simultaneous preparation of several pure aminoacyl-tRNA synthetases . As examples, the large scale purification of phenylalanyl-and valyl-tRNA synthetases are described.

Ann Microbiol (Paris), 1977 Jan, 128A(1), 3 - 18
Yeast protoplasts from stationary and starved cells: preparation, ultrastructure and vacuolar development; Schwencke J et al.; The conversion of stationary and starved yeast cells into protoplasts is described . The method is rapid, simple and can be applied to a variety of stationary yeast cells . Preincubation of yeast cells in the presence of pronase was essential for effective conversion into protoplasts . Baker's yeast and seven defined yeast strains, including one "petite", were studied . All of them were efficiently transformed into protoplasts in 60 to 90 min, depending on the strain culture conditions and the age of the culture . Protoplasts may be obtained even from late-stationary cells which contain spores . Saccharomyces cerevisiae cells subjected to complete starvation conditions in water, could also be completely transformed into protoplasts, even after 48 h of starvation . Electron microscope examination of stationary protoplasts from three different yeast strains showed no evidence of a remaining cell-wall . S . cerevisiae stationary cells show a very developed vacuolar system, a number of "lipid granules" and a few altered mitochondria . Endomycopsis fibuligera and Candida tropicalis stationary protoplasts show a similar fine structure, but "lipid granules" were completely absent.

Biotechnol Bioeng, 1977 Jan, 19(1), 69 - 86
Computer-aided baker's yeast fermentations; Wang HY et al.; The economics of yeast production depend heavily upon the cellular yield coefficient on the carbon source and the volumetric productivity of the process . The application of an on-line computer to maximize these two terms during the fermentation requires a continuous method of measuring cell density and growth rate . Unfortunately, a direct sensor for biomass concentration suitable for use in industrial fermentations is not available . Material balancing, with the aid of on-line computer monitoring, offers an indirect method of measurement . Laboratory results from baker's yeast production in a 14-liter fermentor (with a PDP-11/10 computer for on-line analyses) show this indirect measurement technique to be a viable alternative . From the oxygen uptake and carbon dioxide production data, gas flow rate, and ammonia addition rate, the cell density during the fermentation has been estimated and found to compare well with actual fermentation data.

Z Allg Mikrobiol, 1977, 17(5), 391 - 402
Lysis of intact yeast cells and isolated cell walls by an inducible enzyme system of Arthrobacter GJM-1; Vrsanska M et al.; Bacterium Arthrobacter GJM-1 known in the literature as a good producer of alpha-mannanase was found to accumulate in the culture fluid lytic activities against viable yeast cells during growth on isolated cell walls or beta-glucan fractions of yeast . The accumulation of the lytic activities showed an inducible character . The lytic system produced in the medium containing baker's yeast cell walls was capable of complete solubiliaztion of cell wals in vitro . The system lysed viable cells of a number of yeast species and induced their conversion to protoplasts in an osmotically stabilized medium . The lytic system showed different pH and temperature optima when viable cells or isolated cell walls were used as substrates . The pH optimum of the lysis of isolated cell walls was identical with pH optimum of beta-glucanase activities in the crude system . The results pointed out that in the lysis of intact cells, in addition to beta-glucanases, some other factor is involved . Substantial differences in the nature of the outer and the inner surface of cell walls of Saccharomuces cerevisiae were confirmed in this paper based on the different susceptibility to lysis of the cell walls in vivo and in vitro.

Biotechnol Bioeng, 1977 Jan, 19(1), 27 - 42
A fermentation process for producing both ethanol and lysine-enriched yeast; Tanner RD et al.; In 18 batch-fermentation experiments, baker's yeast was grown in an enriched mineral medium, containing 10% by weight glucose, at various pH and temperature levels . The pH and temperature are just two representative engineering variables which can be easily varied at negligible cost . The commercial yeast inoculum, 20% by weight or about .16% viable cells, was selected to represent industrial (nonsterile) conditions . Free L-lysine, ethanol, and cell growth were followed in time for each batch run held at a fixed pH and temperature . The maximum free lysine level reached at either 10 1/2 or 24 hr occurred at a pH of 5 and 32 degrees C . At 24 hr, the peak free lysine level, 120 mg/liter, is three times as great as the uncontrolled pH counterpart . In terms of total L-lysine (free plus protein-bound) the peak represents a 25% improvement over the uncontrolled case, based on an average 3.5% lysine level per cell weight . The greatest measured cell level, .9% by weight in the fermentation broth, or a 5 1/2-fold increase over th inoculum, was reached during the 36 degrees C and pH 3 run, while the largest measured ethanol value (3%, or 30% conversion by weight from glucose) was achieved during the 28 degrees C and pH 6 experiment . The optimal lysine run product, however, no less than 15% of the maximum cell and 30% of the maximum ethanol levels.

Biochim Biophys Acta, 1976 Dec 1, 454(2), 222 - 9
Purification from baker's yeast of an activator of DNA photolyase; Madden JJ et al.; The activity of purified DNA photolyase from Baker's yeast is enhanced by a compound (Activator (III)) obtained from yeast by chloroform extraction ion exchange chromatography and gel filtration . Thin layer chromatography and spectral data indicate that the compound is homogeneous . Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm . After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that activator (III) contains two separate chromophoric moieties . The chromophore excited by 358 nm light has a pK of 9-11, while the other chromophore has a pK of 4-5, and possibly of 9-11 . The enhancement of photolytic activity by activator (III) at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator with that of heat-denatured photolyase, suggests that the activator may be the chromophore associated with the enzyme.

Biochim Biophys Acta, 1976 Nov 19, 450(2), 165 - 74
Properties of triacylglycerol lipase in a mitochondrial fraction from baker's yeast (Saccharomyces cerevisiae); Schousboe I; A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension . 1 . The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol . The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase . Also parachloromercuribenzoate inhibited the lipase activity . Inhibitory effect of fatty acid was observed at concentrations above 1mM . This inhibition may provide a regulatory mechanism of the lipase in vivo . 2 . On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM . Sonication of the mitochondria increased the activity 2-3 fold . Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin . 3 . The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein . For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when assayed with submitochondrial particles . At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria.

J Assoc Off Anal Chem, 1976 Nov, 59(6), 1344 - 51
Analysis and optimization of a quantitative organic soil neutralization test for disinfectants; Whitmore EJ et al.; An analysis and optimization of a test to quantitatively assess the resistance of disinfectants to neutralization by organic soil is presented . The recommended method uses sterile, dry baker's yeast as a standard organic soil and 24-hr cultures of Staphylococcus aureus and Pseudomonas aeruginosa as test organisms . The test determines the maximum per cent (w/v) of organic soil which a disinfectant can tolerate and remain able to kill about 106 test organisms/ml in 10 min at 25 degrees C . This per cent is defined as the organic soil neutralization number . The procedure offers several advantages over other organic soil capacity tests.

Biochem J, 1976 Nov, 159(2), 363 - 70
Solubilization and other studies on adenylate cyclase of baker's yeast; Varimo K et al.; 1 . Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces . 2 . The enzyme was readily solubilized by Ficoll and by Lubrol PX . Lubrol caused a 2-fold activation . 3 . Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30 degrees C . 4 . The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt . of about 450000.

Hoppe Seylers Z Physiol Chem, 1976 Nov, 357(11), 1605 - 22
Mitochondrial adenosine triphosphatase from yeast, Saccharomyces cerevisiae . Purification, subunit structure and kinetics; Takeshige K et al.; 1 . A procedure for the purification of ATPase extracted by chloroform from baker's yeast (Saccharomyces cerevisiae) is reported . The yield based on submitochondrial particles was 55% and the purification was 100-fold . The isolated complex was homogenous as assessed by gel filtration, ion-exchange chromatography, sedimentation in sucrose gradient and in the analytical ultracentrifuge . The molecular weight determined by gel filtration was 400000 +/- 20000 . Ultracentrifugation yielded s020,w = 12.50 +/- 0.13 S and the laser light scattering study gave a diffusion coeficient of D20w - 2.92 X 10(-7) cm2 s-1 . The amino acid composition as well as absorption, fluorescence, and circular dichroism spectra, from which the helicity of 39% was evaluated, are given . 2 . On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, six components with molecular weights of 58500(alpha), 55000 (beta), 42000, 34000 (gamma), 10000(delta), and 8600 (epsilon) were observed with a stoichiometry of 3:3:1:1:1:1 . The amino acid composition is given for alpha + beta and gamma as well as delta and epsilon components . 3 . The maximum specific activity of the enzyme was 200 U/mg under the optimum conditions . The enzyme was inactivated by incubation at 0 degrees C and strongly inhibited by the antibiotic Dio-9 but not by oligomycin and N, N'-dicyclohexyl-carbodiimide . The effects of kinetic parameters and anions on the enzyme are reported . Two active sites for Mg-ATP with Km values of 0.045mM and 0.37mM and a single activie site for Mg-ITP with Km = 0.179mM were found . A study of the temperature dependence of the maximum activity revealed a straight line in the Arrhenius plots with an activation energy of 11.0 kcal/mol (= 46 kH/mol).

Biotechnol Bioeng, 1976 Oct, 18(10), 1455 - 62
Carbon dioxide inhibition of yeast growth in biomass production; Chen SL et al.; Saccharomyces cerevisiae was grown under aerobic and substrate-limiting conditions for efficient biomass production . Under these conditions, where the sugar substrate was fed incrementally, the growth pattern of the yeast cells was found to be uniform, as indicated by a constant respiratory quotient during the entire growing period . The effect of carbon dioxide was investigated by replacing portions of the nitrogen in the air stream with carbon dioxide, while maintaining the oxygen content at the normal 20% level, so that identical oxygen transfer rate and atmospheric pressure were maintained for all experiments with different partial pressures of carbon dioxide . Inhibition of yeast growth was negligible below 20% CO2 in the aeration mixture . Slight inhibition was noted at the 40% CO2 level and significant inhibition was noted above the 50% CO2 level, corresponding to 1.6 X 10(-2)M of dissolved CO2 in the fermentor broth . High carbon dioxide content in the gas phase also inhibited the fermentation activity of baker's yeast.

Biochim Biophys Acta, 1976 Sep 28, 446(1), 310 - 20
Study of the biological significance of cytochrome methylation . I . Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c; Polastro E et al.; The iso-cytochromes c from baker's yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c . Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters . Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned . This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from baker's yeast . No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from baker's yeast . Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded . Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetes's cytochromes as compared to mammalian ones.

Biochemistry, 1976 Sep 7, 15(18), 4131 - 8
Hydrolytic action of aminoacyl-tRNA synthetases from baker's yeast: "chemical proofreading" preventing acylation of tRNA(I1e) with misactivated valine; von der Haar F et al.; Phe-tRNAPhe-C-C-A, Val-tRNAVal-C-C-A, and Ile-tRNAIle-C-C-A, which accept their amino acid on the 2'-OH of the 3'-terminal adenosine, are hydrolyzed readily by their aminoacyl-tRNA synthetase . If the 3'terminal adenosine in these tRNAs is replaced by either 3'-deoxyadenosine or formycin, little if any hydrolysis can be observed . Correspondingly Ser-tRNASer-C-C-A which accepts serine on the 3'-OH of the 3'-terminal adenosine is hydrolyzed by seryl-tRNA synthetase, whereas Ser-tRNASer-C-C-2'dA and Ser-tRNASer-C-C-F are not . Tyr-tRNATyr-C-C-A and all modified Tyr-tRNATyr-C-C-N, which can accept tyrosine on either the 2'OH or the 3'-OH of the 3'terminal adenosine, are not hydrolyzed by tyrosyl-tRNA synthetase . The data can be rationalized assuming that hydrolysis takes place only if the amino acid is bound to the nonaccepting OH and hence is not positioned at the amino acid binding site upon formation of the complex between aminoacyl-tRNA and aminoacyl-tRNA synthetase . In the formycin-carrying tRNA, the amino acid bound to the nonaccepting OH seems to be inaccessible to the enzymatic groups responsible for hydrolysis . Val-tRNAIle-C-C-3'dA and Ile-tRNAIle-C-C-3'DA cannot be hydrolyzed by isoleucyl-tRNA synthetase . Val-tRNAIle-C-C-A is hydrolyzed by the enzyme five times more rapidly than Ile-tRNAIle-C-C-A . Whereas Ile-tRNAIle-C-C-F is absolutely stable, Val-tRNAIle-C-C-F si hydrolyzed immediately . As shown by the earlier finding that valine misactivated by isoleucyl-tRNA synthetase cannot be permanently transferred to tRNAIle-C-C-A but to tRNAIle-C-C-3'dA, the 3'-OH is essential for preventing transfer of misactivated valine . It thus appears that valine is hydrolyzed off Val-tRNAIle-C-C-N if it is bound to the accepting 2'-OH in the binding site for isoleucine . A hypothesis is offered attempting to explain the experimental observations in mechanistic terms . We consider the hydrolytic action of the aminoacyl-tRNA synthetases as a general mechanism of "chemical proofreading" in the protein biosynthesis.

Biochem J, 1976 Sep 1, 157(3), 773 - 5
Iso-cytochrome c species from baker's yeast . Analysis of their circular-dichroism spectra; Looze Y et al.; The circular-dichroism spectra of baker's-yeast iso-1- (methylated and unmethylated forms) and iso-2-cytochrome c species were examined between 200 and 600nm . In the visible region the yeast haemoproteins have characteristics nearly indistinguishable from those of horse heart cytochrome c . From the spectra in the u.v . region the latter appears, however, to be more helical . It is proposed that the likely element of non-helical structure in iso-1-cytochrome c is residues 62-70.

Mol Cell Biochem, 1976 Aug 30, 12(2), 73 - 9
Some properties of the adenosine triphosphatase systems of two yeast species, Saccharomyces cerevisiae and Rhodotorula glutinis; Sigler K et al.; 1 . Total ATPase levels were determined in homogenate fractions of baker's yeast, Saccharomyces cerevisiae K and Rhodotorula glutinis . The maximum ATPase activities in 8000 X g supernatant of the three yeast strains were 6.0, 1.9, and 2.2 mmol Pih-1 (gDS)-1, respectively; the activities in the sediment were somewhat higher . Exponential cells of S . cerevisiae K and R . glutinis exhibited higher ATPase levels than did the stationary cells . 2 . The total ATPase activity in both yeast species showed a maximum at ph 6.8 a minimum at pH 7.2, and another broader masimum around pH 8.0 . 3 . No significant NaK-ATPase activity was detected in baker's yeast, in either the exponential or the stationary cells of R . glutinis, and in exponential S . cerevisiae K cells in the pH range of 6.0-9.3 . 4 . Stationary cells of S . cerevisiae K exhibited, at pH 7.0-8.5, A Na,K-ATPase activity attaining 9% of total ATPase level . 5.3 X 10(-3) M phenylmethyl sulphonyl fluoride had no effect on the total ATPase level in S . cerevisiae and inhibited the activity in R . glutinis by 25%; it did not bring forth any Na,K-ATPase activity apart from that found in its absence . 6 . 1.5 M urea lowered the ATPase activity in R . glutinis by 68% but had no effect on S . cerevisiae cells . 10(-5) M dicyclohexylcarbodiimide suppressed the ATPase activity in S . cerevisiae and R . glutinis by 74 and 79%, respectively . Neither agent revealed and additional Na,K-ATPase activity . 7 . The comparison of Na,K-ATPase activities with data on K+ fluxes across the yeast plasma membrane suggested that even with the lower flux values the Na,K-ATPase, even if present, would account for a mere 40% of transported ions . The results imply that the active ion transport in yeasts is energized by mechanisms other than the Na,K-ATPase.

Biokhimiia, 1976 Aug, 41(8), 1426 - 34
{Some peculiarities of transfer RNAs hydrolysis by exonuclease A5}; Lbvova TN et al.; Unusual kinetics of the hydrolysis of the baker's yeast total tRNA and tRNA1Val by exonuclease A5 was found (two stages of the reaction; low initial velocity and still lower velocity of the second reaction stage; high Km values) . These peculiarities were shown to be due to the three-dimensional structure of the substrate, which makes a large part of the tRNA molecule resistant to the exonuclease A5 attack . The exhaustive tRNA hydrolysis observed in the presence of very large doses of exonuclease A5 may be explained by a slight (less than 1%) endo-RNAase contamination in the exonuclease A5 preparation . The hydrolysis conditions become optimal, i.e . the exhaustive hydrolysis proceeds most rapidly, when the amount of endo-RNAase admixture reaches 20--30% . The artificial enzyme mixture of such a composition is a convenient reagent for RNA and expecially for tRNA hydrolysis under mild conditions to 5'-mononucleotides . The considerable resistance of tRNA to pure exonuclease A5 action will make it possible porbably to use tRNA instead of circular nucleic acids for endonuclease admixtures estimation in enzyme preparations.

Eur J Biochem, 1976 Aug 1, 67(1), 215 - 21
Affinity labelling of tRNA nucleotidyltransferase from baker's yeast with tRNAPhe modified on the 3'-terminus; Sternbach H et al.; 2'-Deoxy-2'-amino-cytidylic acid can be incorporated into position 75 of tRNAPhe from yeast by tRNA nucleotidyltransferase yielding tRNAPhe-C-C(2'NH2) . tRNAPhe-C-C(2'NH2) can be reacted with the N-hydroxysuccinimide esters of bromoacetic acid of mercuriacetic acid to yield the derivatives tRNAPhe-C-C(2'NHCOCH2Br) and tRNAPhe-C-C(2'NHCOCH2Hg+OH-) . Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics . The enzyme can be protected against inhibition by its substrates tRNAPhe-C and tRNAPhe-C-C as well as ATP and CTP . A covalent, isolatable 1:1 complex between tRNAPhe-C-C(2'NHCOCH2Br) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p-hydroxymercuribenzoate.

Fed Proc, 1976 Aug, 35(10), 2124 - 30
Do evolutionary changes in cytochrome c structure reflect functional adaptations?
Margoliash E, Ferguson-Miller S, Kang CH, Brautigan DL.
Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted . Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components . However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena . Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase . The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP . The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control . The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.

Biochem J, 1976 Aug 1, 157(2), 389 - 93
Kinetic mechanism from steady-state kinetics of the reaction catalysed by baker's-yeast glucose 6-phosphate dehydrogenase in solution and covalently attached to sepharose; Gould BJ et al.; 1 . The reaction catalysed by glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast was studied in 42mM-glycylglycine buffer, pH7.4 at 25 degrees C, by initial-velocity studies and by the use of NADPH as a product inhibitor . 2 . The reactions catalysed by both the soluble enzyme and a stable enzyme covalently attached to CNBr-activated Sepharose 4B probably follow an ordered reaction mechanism with NADP+ and NADPH as the leading reactants . 3 . The kinetic constants obtained for the soluble enzyme lere: KNADP+m, 19 muM; KNADP+s, 23 muM; KNADPHs, 15 muM . Similar values were obtained for the immobilized enzyme . 4 . The assay of the immobilized enzyme was done by using a micro packed-bed recirculation reactor, and the advantages of this technique are discussed.

Biochem J, 1976 Aug 1, 157(2), 289 - 94
Preparation of immobilized baker's-yeast glucose 6-phosphate dehydrogenase attached to modified sepharose and sephadex and a comparison of the properties of these preparations with those of the soluble enzyme; Goheer MA et al.; 1 . Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity . This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C . 2 . Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme . 3 . These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C . The soluble enzyme is inactivated by these conditions . 4 . The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively . The three immobilized-enzyme preparations were completely stable over this entire pH range . 5 . The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively . The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6 . Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.

Eur J Biochem, 1976 Jul 15, 66(3), 493 - 7
Valyl-tRNA, isoleucyl-tRNA and tyrosyl-tRNA synthetase from baker's yeast . Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction; Freist W et al.; Nineteen analogs of ATP have been tested in the aminoacylation of valyl-tRNA, isoleucyl tRNA and tyrosyl-tRNA synthetases from baker's yeast . Four compounds are substrates for valyl tRNA and two for isoleucyl-tRNA synthetase, but there is no modified substrate for the tyrosyl tRNA synthetase . There is one inhibitor for valyl-tRNA synthetase, eight compounds inhibit isoleucyl-tRNA synthetase and two compounds inhibit tyrosyl-tRNA synthetase . Their Km and Ki and V values have been determined . The substrate specificity shows that positions 2, 6, 7, 8, 9, 2', and 3' of ATP are important for catalytic action of these aminoacyl-tRNA synthetases.

Eur J Biochem, 1976 Jul 15, 66(3), 523 - 33
Subunit structure and multifunctional properties of yeast phosphoglyceromutase; Sasaki R et al.; 1 . A new and efficient method for preparation of pure phosphoglyceromutase from baker's yeast (Saccharomyces cerevisiae) is described . Proteolytic alterations of the enzyme during extraction can be minimized by grinding the dried yeast with aluminium oxide at low temperature . 2 . Yeast phosphoglyceromutase contains four highly similar, probably idential subunits of molecular weight 28000, a conclusion based on the following observations . Polyacrylamide gel electrophoresis containing dodecylsulphate or urea gives a single band, indicating that the enzyme is composed of four subunits similar in their molecular weight and net charge . Cyanogen bromide cleavage and tryptic digestion of the enzyme yield the number of peptides expected for identical subunites from the amino acid composition analysis . 3 . The purified phosphoglyceromutase preparation has bisphosphoglyceromutase activity synthesizing 2,3-bisphosphoglycerate from 1,3-bisphosphoglycerate and 3-phosphoglycerate . It has been reported that yeast phosphoglyceromutase catalyzes the hydrolysis of 2,3-bisphosphoglycerate at the same active site which catalyzes the phosphoglyceromutase reaction {Sasaki, R . et al (1971) Biochim . Biophys, Acta, 227, 584-594, 595-607} . Immunological studies and chemical modification experiments indicate that bisphosphoglyceromutase activity also is due to the phosphoglyceromutase protein and involves amino groups which have been shown to be essential for the other two activities.

Mikrobiologiia, 1976 JUL-AUG, 45(4), 646 - 9
{Effect of the physiological properties of yeast strains on their formation of higher alcohols and aldehydes and on the composition of nitrogen compounds in the fermented wort}; Gracheva IM et al.; Dynamics of formation of by-products was studied with the following yeast races: Sacch . cerevisiae, strain Odesskaya-14 (baker's yeast); Sacch . vini, strain Prikumskaya 80/9 (wine yeast) . The yeast cultures were found to be very similar by the rate of biomass accumulation, ethanol production, and the fractional composition of nitrogen compounds . The concentration of accumulated higher alcohols depended on the mass of yeast cells, their growth rate, and the duration of cultivation.

Biofizika, 1976 Jul-Aug, 21(4), 648 - 52
{New approach to the interpretation of vibrational spectra of microbial cells}; Zaslavskii BIu et al.; IR-technique was applied to study baker's yeast cell and its envelope fragments . The quantitative evaluation of spectra was made using the polysaccharide band at 1070 cm-1 as the bench one . The comparison of the spectra of the whole cells and of the envelope fragments suggested that the spectrum of the yeast cell was governed by the composition of its envelope only . ATR-spectra of the whole cells and of envelope debris were obtained and the comparison of these with the "absorption" ones made it possible to conclude that the yeast cells vibration spectrum was for the most part the reflection spectrum governed by the composition of the cell envelope rather than the absorption one.

Biotechnol Bioeng, 1976 Jul, 18(7), 975 - 86
Large-scale disintegration of microorganisms by freeze-pressing; Magnusson KE et al.; A semicontinuous press has been constructed for the disintegration of microorganisms and other biological material by freeze-pressing, i.e., pressure extrusion of frozen material through a narrow hole . The material to be freeze-pressed is frozen in the form of cylindrical rods, which fit into the pressure chamber and are extruded by a piston forced back and forth by means of a hydraulic pump . At a sample temperature of -35 degrees C and a press temperature of -20 degrees C, about 90% disruption is achieved in a single passage of undiluted baker's yeast (Saccharomyces cerevisiae, 270 mg/g) through the orifice of the pressure chamber . With this press about 10 kg of material can be freeze-pressed per hour.

Biotechnol Bioeng, 1976 Jul, 18(7), 1001 - 16
Fed batch culture of Saccharomyces cerevisiae: a perspective of computer control to enhance the productivity in baker's yeast cultivation; Aiba S et al.; A means to avoid the glucose effect in the production of baker's yeast from glucose and/or molasses in a fed batch culture by controlling the feed rate of fresh medium with an ad hoc measurement of the respiratory quotient, RQ, is presented . The feed rate is changed stepwise here such that the value of RQ ranges from 1.0 to 1.2 throughout the cultivation . Thus far, the specific growth rate based on the total cell mass and the growth yield obtained throughout are 0.24 hr-1 and 0.55 g cell/g glucose . Prior to the experimental run mentioned above, equations to predetermine the feed rate and concentration of glucose in the feed are derived from the mass balance of limiting substrates (glucose) . Since values of either RQ or Io2 (Qo2 x, oxygen consumption rate with respect to the total cell mass in the fermenter) can be measured quite easily and reliably, computer control of the fermentation in light of this information is discussed.

Eur J Biochem, 1976 Jun 1, 65(2), 537 - 42
Baker's yeast flavocytochrome b2 (L-(+)-lactate dehydrogenase) . An immunological study of structure and function; Guiard B et al.; Baker's yeast flavocytochrome b2 has been suggested before to be a polyglobular protein formed after a gene fusion process . It is known that one of the potential globules, the heme-binding core, can exist in the absence of the rest of the protein . Using antibodies elicited against the whole enzyme and against the core, we show in this paper that part of the core surface, and in particular the mouth of the heme-binding crevice, must be exposed in the complete enzyme molecule . Antibodies inhibit the activity of intact and proteolytically-cleaved enzymes, which normally show a number of differences in some kinetic parameters . Interestingly, antibodies against the core induce a modification of some kinetic constants for the cleaved enzyme (in particular the Km for the substrate and Ki for D-(-)-lactate, bringing them back to values similar to those for the intact enzyme . These results can be interpreted as a tightening of the cleaved enzyme by anti-core antibodies . The conformational effect is transmitted from the heme-binding region to other parts of the molecule . This implies some intimate contacts between the core and the rest of the protein.

Hoppe Seylers Z Physiol Chem, 1976 Jun, 357(6), 819 - 23
Phenylalanyl-tRNA synthetase from baker's yeast: specificity and quantitation of affinity elution with tRNA; von der Haar; TRNAPhe is able to elute phenylalanyl-tRNA synthetase from cation exchangers in a 1:1 ratio . Elution of phenylalanyl-tRNA synthetase in a 1:1 ratio is also observed for four noncognate tRNAs investigated, specific for valine, serine, isoleucine and tyrosine . If protein mixtures are subjected to affinity elution the cognate pair {tRNAPhe-phenylalanyl-tRNA synthetase} is eluted first, followed by noncognate pairs . The unspecific elution is not influenced by complexation of phenylalanyl-tRNA synthetase with an analog of phenylalanyl-adenylate.

J Clin Invest, 1976 Jun, 57(6), 1635 - 43
Hereditary deficiency of the fifth component of complement in man . II . Biological properties of C5-deficient human serum; Rosenfeld SI et al.; The first known human kindred with hereditary deficiency of the fifth component of complement (C5) was documented in the accompanying report . This study examines several biological properties of C5-deficient (C5D) human serum, particularly sera obtained from two C5D homozygotes . The proband, who has inactive systemic lupus erythematosus is completely lacking C5, while her healthy half-sister has 1-2% of normal levels . Both sera were severely impaired in their ability to generate chemotactic activity for normal human neutrophils upon incubation with aggregated human gamma-globulin or Escherichia coli endotoxin . This function was fully restored in the sibling's serum, and substantially improved in the proband's serum, by addition of highly purified human C5 to normal serum concentrations . Sera from eight family members who were apparently heterozygous for C5 deficiency gave normal chemotactic scores . The ability of C5D serum to opsonize Saccharomyces cerevisiae (baker's yeast) or Candida albicans for ingestion by normal neutrophils was completely normal . In addition, C5D serum was capable of promoting normal phagocytosis and intracellular killing of Staphylococcus aureus . The proband's serum was incapable of mediating lysis of erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria in both the sucrose hemolysia and acid hemolysis tests, and also lacked bactericidal activity against sensitized or unsensitized Salmonella typhi . The sibling's serum, containing only 1-2% of normal C5, effectively lysed S . typhi, but only at eightfold lower serum dilutions as compared to normals . These findings underscore the critical role of C5 in the generation of chemotactic activity and in cytolytic reactions, as opposed to a nonobligatory or minimal role in opsonization, at least for the organisms under study.

Biotechnol Bioeng, 1976 Jun, 18(6), 865 - 83
Influence of cell concentration, temperature, and press performance on flow characteristics and disintegration in the freeze-pressing of Saccharomyces cerevisiae with the X-press; Magnusson KE et al.; The pressure required for initiation of flow when freeze-pressing with the X-press is related to the phase boundaries of water, particularly those between ice I and liquid even at temperatures around -25 degrees C and lower . Widening the orifice of the pressure chamber to diameters larger than 2.5 mm leads to lower pressures and less extensive cell disintegration . Pressing Saccharomyces cerevisiae slowly with the aid of a manual hydraulic jack at -25 degrees C produces a disintegration of 60-75% irrespective of cell concentration . Pressing at -35 degrees C shows no clear differences . Pressing more rapidly with the aid of a motor-driven hydraulic press produces a similar extent of disruption of diluted cell suspensions (5.4 mg/g) as slow pressing . However, freeze-pressing a paste of baker's yeast (270 mg/g) increases the degree of disintegration . Under these conditions the disintegration is further enhanced by a lower temperature, -35 degrees C, and by a high velocity of flow through the orifice, such that more than 95% of the S . cerevisiae is disrupted by one pressing at less than 2 X 10(8) Pa . Mechanisms for flow through the X-press are suggested and discussed in relation to the phase diagram of water.

Eur J Biochem, 1976 May 17, 65(1), 177 - 82
Arginyl-tRNA synthetase from baker's yeast . Purification and some properties; Gangloff J et al.; Arginyl-tRNA synthetase from baker's yeast (Saccharomyces cerevisiae, strain 836) was obtained pure by a large-scale preparative method, which involves four chromatographic columns and one preparative polyacrylamide gel electrophoretic step . The enzyme has a high specific activity (9000 U/mg) and consists of a single polypeptide chain of molecular weight approximately 73000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate . Amino acid analysis of the enzyme permitted calculation of the absorption coefficient of arginyl-tRNA synthetase (A(1 mg/ml 280 nm)=1.26) . Concerning kinetic parameters of the enzyme we found the following Km values: 0.28 muM, 300 muM, 1.5 muM for tRNA(Arg III), ATP and arginine in the aminoacylation reaction, and 1400 muM, 2.5 muM, and 50 muM for ATP, arginine and PP(i) in the ATP-PP(i) exchange reaction . Arginyl-tRNA synthetase required tRNA(Arg III) to catalyse the ATP-PP(i) exchange reaction.

J Gen Microbiol, 1976 May, 94(1), 180 - 92
Isolation and composition of an alkali-soluble glucan from the cell walls of Saccharomyces cerevisiae; Fleet GH et al.; An alkali-soluble glucan was obtained from the cell walls of Saccharomyces cerevisiae NCYC1109 and baker's yeast by extraction with cold, dilute sodium hydroxide under nitrogen . The glucan, which represented approximately 20% of purified glucan was homogeneous and was shown to be free from contamination by other cell-wall polysaccharides by ultracentrifuging, gel filtration and electrophoresis . In addition to glucose, the glucan contained traces of mannose and nitrogen, but no hexosamine . Structural analyses revealed the presence of 80-85% (1 leads to 3)-beta-D linkages, 8-12% (1 leads to 6)-beta-D linkages and 3-4% branched residues linked through C-1, C-3 and C-6 . The molecular weight of the glucan was estimated to be about 250000 . Electron-microscopic examination of the cell walls after alkali extraction showed that an amorphous surface layer had been removed revealing numerous bud scar structures.

Eur J Biochem, 1976 May 1, 64(2), 395 - 8
Phenylalanyl-tRNA synthetase from baker's yeast . Salt dependence of steady-state kinetics indicates two molecular forms of the enzyme; von der Haar F; Steady-state kinetic data of aminoacylation of tRNAPhe by phenylalanyl-tRNA synthetase depend on salt concentration . At 5 mM KCl and 20 mM MgSO4 a non-linear curve is found in the double-reciprocal plot for ATP and phenylalanine, while at 200 mM KCl and 50 mM MgSO4 a linear curve is observed . KCl and MgSO4 dependence of the reaction also show biphasic curves with intersection points of the two extrapolated linear parts at 50 mM and 10 mM, respectively . A biphasic curve is also found if the concentration of CTP is varied at constant low ATP concentration . Extrapolations of the linear parts of the curves for ATP as well as for CTP at 5 mM KCl and 20 mM MgSO4 intersected the 1/{NTP} axis at 1.2 +/- 0.1 mM . Hence the existence of a non-linear curve for ATP as well as phenylalanine does not necessarily indicate two non-equivalent binding sites for these substrates . A more likely explanation is the existence of two different molecular forms of phenylalanyl-tRNA synthetase which are interconvertible by salt . This explanation is substantiated by the observation that proteolytic digestion of phenylalanyl-tRNA synthetase is more easily achieved at low than at medium ionic strength . In addition mischarging of tRNAIle with phenylalanine by phenylalanyl-tRNA synthetase occurs at a moderate rate at 5 mM KCl and 20 mM MgSO4 whereas it is largely depressed by addition of either 5 mM CTP or 150 mM KCl.

Eur J Biochem, 1976 May 1, 64(2), 389 - 93
Phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast . Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction; Freist W et al.; Eighteen analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast . Four compounds are substrates for phenylalanyl-tRNA synthetase, five for seryl-tRNA synthetase, one compound is an inhibitor for both enzymes; their Km and Ki and V values have been determined . The substrate specificity shows that for the catalytic action of both enzymes with these substrates positions 6, 7, 8 and 9 of the purine moiety and positions 2' and 3' of the ribose moiety are important.

Proc Natl Acad Sci U S A, 1976 May, 73(5), 1471 - 5
Functional genetic expression of eukaryotic DNA in Escherichia coli; Struhl K et al.; We have isolated a segment of DNA from the eukaryote Saccharomyces cerevisiae (baker's yeast) as a viable molecular hybrid of bacteriophage lambda DNA which, when integrated into the chromosome of an E . coli histidine auxotroph, allows this bacterium to grow in the absence of histidine . The nonrevertable, histidine auxotroph lacks the enzymatic activity of imidazole glycerol phosphate (IGP) dehydratase (EC 4.2.1.19) . From genetic experiments, we conclude that expression of the segment of yeast DNA results in the production of a diffusible substance and that transcription necessary for the complementation is most likely initiated from the segment of eukaryotic DNA.

Biochim Biophys Acta, 1976 Mar 26, 424(3), 366 - 75
Triacylglycerol lipase activity in baker's yeast (Saccharomyces cerevisiae); Schousboe I; An investigation of intracellular triacylglycerol lipase activity in baker's yeast (Saccharomyces cerevisiae) has been performed using emulsified triolein as substrate . Bovine serum has been used as emulsifier since it was found superior to gum arabic and albumin with respect to reproducibility of both triacylglycerol concentration in the assay mixture and specific lipase activity . No extracellular activity could be detected neither with whole cells nor with water or detergent "extracts" of intact cells as enzyme source . With disrupted cells the level of triacylglycerol lipase activity at a triacylglycerol concentration of 9.6 mM, at pH 7.5, and 30 degrees C was 190 mumol free fatty acids per h per g disrupted cells . Fractionation of a cytoplasmic extract of disrupted cells revealed that about 70% of the activity was associated with membrane fractions and 60% of this activity was present in the mitochondrial fraction . Purification of this fraction was followed by an increase in specific lipase activity which parallels the increase in specific activity of the cytochrome c oxidase.

Biochim Biophys Acta, 1976 Mar 11, 429(1), 274 - 82
Purification of protoplast-secreted acid phosphatase from baker's yeast . Effect on adenosine triphosphatase activity; Mildner P et al.; In order to examine acid phosphatase (EC 3.1.3.2) and ATPase (EC 3.6.1.3) activities of baker's yeast (pH optimum 3.5) a protoplast-secreted enzyme preparation was purified and some physical and chemical properties were studied . Three protein fractions containing ATPase and acid phosphatase activities, in the same ratio as the initial preparation, were separated by ion-exchange chromatography . The first fraction which had the highest protein content yielded a homogeneous preparation after Sepharose 4B chromatography and was used in further studies . An attempt to estimate molecular weight of this protein was made . Attempts to separate acid phosphatase and ATPase activities by ion-exchange chromatography, gel filtration, isoelectric focusing and sucrose density gradient centrifugation have been unsuccessful . Both activities behaved the same way to heat and urea denaturation . These results suggest that the two activities are associated with the same protein molecule.

Biochem J, 1976 Feb 1, 153(2), 447 - 54
The nucleotide sequence of cysteine transfer ribonucleic acid from baker's yeast . Identification of the products from partial degradation of the molecule and derivation of the complete sequence; Holness NJ et al.; 1 . A series of large oligonucleotide fragments derived from tRNA Cys, were separated chromatographically and the sequence of each was deduced by examination of the products of digestion with pancreatic and T1 ribonucleases . 2 . The location of the specific cleavage points in the nucleotide chain was similar to that produced by brief treatment with pancreatic ribonuclease . 3 . The fragments could be arranged into two alternative sequences . The correct sequence was deduced by the sequential removal and identification of the first nine nucleotides from the 3'-end of the terminal half of the molecules.

Biochem J, 1976 Feb 1, 153(2), 437 - 46
The nucleotide sequence of cysteine transfer ribonucleic acid from baker's yeast . Products of complete digestion with pancreatic ribonuclease and ribonuclease T1; Holness NJ et al.; 1.The nucleotide chain of tRNA Cys from baker's yeast was readily split at the anticolon into two large fragments by brief treatment with ribonuclease T1.2 . The whole molecule and the two derived large fragments were completely digested with (a) pancreatic ribonuclease and (b) ribonuclease T1 . The fragments present in each of the digests were separated and sequenced by conventional methods . 3 . The groups of fragments derived from the two methods of digestion were entirely compatible with each other . 4 . The molecule is 75 nucleotides long, but, as isolated, lacks the terminal adenosine and the neighboring cytidylic acid residue . The minor nucleotides 1-methyladenylic acid, 7-methylguanylic acid, 5-methylcytidylic acid and N6 (gamma gamma-dimethylallyl)adenylic acid (isopentenyladenylic acid) were identified.

Biochem J, 1976 Feb 1, 153(2), 429 - 35
The extraction and purification of a cysteine transfer ribonucleic acid from baker's yeast; Holness NJ et al.; 1 . A modification of the RPC 1 system of A.D . Kelmers, G.D . Novelli & M.P . Stulberg (1965) (J . Biol . Chem . 240, 3979-3983) is described in which the support medium is a Celite of narrow range particle size treated with dichlorodimethylsilane . 2 . By using this system an apparently pure preparation of tRNA Cys was isolated from baker's yeast tRNA . 3 . This preparation accepted at least 60% of the theoretical quantity of {3-14C}cysteine in a conventional assay and failed to accept isoleucine, phenylalanine, proline, serine or tyrosine . 4 . A theoretical countercurrent-distribution curve calculated by assuming a distribution coefficient K of 2.03 was in excellent agreement with the profiles of E260 and cysteine-acceptor ability after 537 transfers in the 1.85 M-phosphate/formamide/propan-2-ol system of C.M . Connelly & B.P . Doctor (1965) (J . Biol . Chem . 241, 715-719) . 5 . Chromatography of tRNA Cys on Bio-Gel P100 polyacrylamide beads afforded two components one of which was far less efficient than the other in accepting cysteine . The base compositions of the two were similar.

Biochem J, 1976 Feb 1, 153(2), 309 - 19
Some properties of an alcohol dehydrogenase partially purified from baker's yeast grown without added zinc; Dickenson CJ et al.; Alcohol dehydrogenase was partially purified from yeast (Saccharomyces cerevisiae) grown in the presence of 20 muM-MnSO4 without added Zn2+ and from yeast grown in the presence of 1.8 muM-MnSO4 . The enzyme from yeast grown with added Zn2+ has the same properties as the crystalline enzyme from commercial supplies of baker's yeast . The enzyme from yeast grown without added An2+ has quite different properties . It has a mol.wt . in the region of 72000 and an S 20 w of 5.8S . The values can be compared with a mol.wt . of 141000 and an S 20 w of 7.6S for the crystalline enzyme . ADP-ribose, a common impurity in commercial samples of NAD+, is a potent competitive inhibitor of the new enzyme (K1 = 0.5 muM), but is not so for the crystalline enzyme . The observed maximum rate of ethanol oxidation at pH 7.05 and 25 degrees C was decreased 12-fold by the presence of 0.06 mol of inhibitor/mol of NAD+ when using the enzyme from Zn2+-deficient yeast, but with crystalline enzyme the maximum rate was essentially unchanged by this concentration of inhibitor . The kinetic characteristics for the two enzymes with ethanol, butan-1-ol, acetaldehyde and butyraldehyde as substrates are markedly different . These kinetic differences are discussed in relation to the mechanism of catalysis for the enzyme from Zn2+-deficient yeast.

Acta Chem Scand B, 1976, 30(3), 228 - 34
Characterization of charge isomers of yeast phosphoglycerate kinase . Evidence for intracellular differences; Arvidsson L et al.; Three electrophoretic components of phosphoglycerate kinase have been isolated from baker's yeast . The isoionic point of the major component is 7.18 at 10 degrees C . Corresponding values for the minor ones are 6.91 and 7.48, respectively . There is a difference of one charge-unit between the isomers 1 and 2, and between the isomers 2 and 3 . The release of component 3 from the yeast cells appears in contrast to the isomers 1 and 2 to be promoted by an organic solvent, thus suggesting this component to be bound to the cell-membrane . The amino-terminal amino acid residue appears to be N-acetylated serine in each of the three cases . The carboxyl-terminal ends seem to be identical also with -(Ala, Leu, Val, Lys)- Ala-Lys as the ultimate sequence . From the circular dichroism spectra the contents of alpha-helix and beta-structure were estimated to 15 and 40-50%, respectively . Factors have been determined for transformation and comparison of the specific activities as determined under the various conditions used at different laboratories.

Mol Biol (Mosk), 1976 Jan-Feb, 10(1), 142 - 8
{Several structural aspects of the interaction of yeast hexokinase with insulin}; Rubenchik AI et al.; It has been noted that due to formation of the complex with insuline light and hear resistance of hexokinase from baker's yeast rises . Variation of luminescence parameters shows structural modification of enzyme upon complex formation . On the basis of comparison the photoinactivation data and hexokinase photolysis conclusion has been drawn that the tryptophanyl residues are not directly involved in the enzyme active site, although play an important role in supporting the specific structure.

J Nutr Sci Vitaminol (Tokyo), 1976, 22(1), 53 - 61
Effect of reductones on glyoxalase I1; Iio M et al.; The effect of some reductones on glyoxalase I prepared from animal and microbial origins has been studied . The enzyme was extracted from ox liver or baker's yeast and partially purified by ammonium sulfate fractionation, gel filtration and ion exchange chromatography . Aliphatic reductones such as ascorbic acid, ascorbic acid 3-phosphate and triose reductone showed strong to medium inhibition, while dehydroascorbic acid showed no inhibition . Kinetic analysis indicated that the inhibition mechanism of ascorbic acid was uncompetitive . Varying extents of inhibition were observed among three kinds of diphenols belonging to aromatic reductones . They were in the order of increasing inhibitory power resorcinol, hydroquinone and catechol for the ox liver enzyme, and catechol, resorcinol and hydroquinone for the yeast enzyme . p-Benzoquinone, an oxidized reductone, exhibited marked inhibition on both enzymes . Its action seemed due to reaction with amino and/or sulfhydryl functions of enzyme protein and those of glutathione, one of the substrates.

Folia Microbiol (Praha), 1976, 21(2), 125 - 30
Loss of inducible D-galactose transport by baker's yeast after osmotic treatment; Horak J et al.; The ability of Saccharomyces cerevisiae to transport D-galactose and related sugars with an axial hydroxyl group at C-4, acquired by induction with D-galactose, was lost either by exposing early exponential-phase cells to an osmotic shock involving incubation in 0.6M NaC1O4, 0.66M sucrose and 1mM histidine and transfer to 5mM Tris-HC1 with 2mM dithiothreitol, or simply by transferring them to distilled water . The total amount of protein thus released was 0.1--0.35 and 0.1 mg per mg dry wt., respectively . The shock fluid contained at least six proteins, among them a galactose-binding component . L-Arabinose transport could not be restored by adding the concentrated shock fluid to depleted cells but cells remained viable after the shock and resynthesized the transport system if incubated in a galactose-containing growth medium.

Int Arch Allergy Appl Immunol, 1976, 50(6), 751 - 4
Anaphylactoid responses of two types of genetically different rats to horse serum and yeast; Cooper CA et al.; Rats selectively bred for non-reactivity to clinical dextran (NR rats) fail to exhibit the anaphylactoid reaction to systemic baker's yeast or horse serum . Rats which respond with an anaphylactoid reaction to clinical dextran (R rats) react to baker's yeast but do not react to horse serum . The widespread oedema produced in R rats by systemic baker's yeast provides yet another clear method of differentiating them from NR rats.

Proc Soc Exp Biol Med, 1976 Jan, 151(1), 72 - 7
The effects of salicylic acid on metabolism and potassium ion content in yeast; Scharff TG et al.; Under anaerobic conditions, at low pH and 30 degrees, commercial baker's yeast loses K+ ion in the presence of salicylic acid . Glucose utilization is inhibited . In suspensions containing no glucose, carbohydrate stores of the cell are dissimilated to carbon dioxide and alcohol . The ion loss and inhibitory effects of salicylic acid on glucose utilization are reversed by washing the cells free of salicylate . The loss of K+ appears to be due at least partly to a K+-H+ exchange process . An unexplained maximum is seen in the curves of either net K+ loss or K+ efflux versus salicylic acid concentration . At 6 degrees the effects of salicylic acid on both endogenous metabolism and net K+ loss are minimal . Furthermore, no maximum is seen in the K+ loss-salicyclic concentration curve at this temperature . It is generalized that salicylic acid or salicylate may elicit K+ leakage from many types of cells, i.e., a fundamental action of this compound may be its ability to affect (reduce) K+ content of the cell; furthermore, it appears that the salicylate effects on K+ loss may be associated in an as-yet-unknown manner with the metabolic effects of this compound . The effects of salicylate on K+ loss in yeast may not be unique for this compound, since no experiments of this nature have been done with other penetrating undissociated acids.

Arch Microbiol, 1975 Dec 31, 106(3), 271 - 3
Effect of aeration on the activity of gluconeogenetic enzymes in Saccharomyces cerevisiae growing under glucose limitation; Haarasilta S et al.; 1 . The effect of aeration on the key enzymes of gluconeogenesis was studied in baker's yeast (Saccharomyces cerevisiae) and in a nonrespiratory variant of S . cerevisiae grown under glucose limitation . 2 . In baker's yeast phosphoenolpyruvate carboxykinase, hexosediphophatase and isocitrate lyase were completely repressed under anaerobic conditions . Their repression could be partially reversed by using intense aeration . 3 . In the nonrespiratory variant these enzymes were absent independently of aeration . 4 . Pyruvate carboxylase of baker's yeast showed maximal activity under anaerobic conditions . In the nonrespiratory variant pyruvate carboxylase had low activity under both anaerobic and aerobic conditions.

Biochim Biophys Acta, 1975 Dec 18, 410(2), 318 - 32
Glucanases in Schizosaccharomyces . Isolation and properties of an exo-beta-glucanase from the cell extracts and culture fluid of Schizosaccharomyces japonicus var . versatilis; Fleet GH et al.; (11 Cell extracts and extracellular culture fluids of species of the yeast genus Schizosaccharomyces exhibited exo-beta-(1 leads to 3)- and exo-beta-(1 leads to 6)-glucanase (EC 3.2.1.-) activities . (2) Using a combination of Sephadex G-100 and DEAE-cellulose chromatography, the exo-beta-(1 leads to 3)-glucanases from the cell extracts and culture fluid of Schizosaccharomyces japonicus var . versatilis were purified extensively . The enzymes from either location exhibited similar purification and other properties . (3) The purified enzymes hydrolysed the beta-(1 leads to 6)-glucosidic linkage in addition to the beta-(1 leads to 3) linkage . Heat denaturation, inhibition and electrophoretic studies indicated that both hydrolytic activities were properties of a single protein . Laminarin and pustulan hydrolysis followed Michaelis-Menten kinetics . The Km and V for laminarin hydrolysis were 6.25 mg/ml and 350 mumol of glucose released/min/mg protein, and for pustulan they were 166 mg/ml and 52 mumol of glucose released/min/mg protein . (4) The exo-beta-glucanase was assigned a molecular weight of 43 000 . (5) the purified enzyme failed to hydrolyse isolated cell walls from either baker's yeast or Schizosaccharomyces pombe or to induce protoplast formation from intact cells of S . japonicus var . versatilis or Saccharomyces cerevisiae.

Radiat Environ Biophys, 1975 Dec 4, 12(4), 281 - 90
Studies on the energy metabolism during the respiratory process by baker's yeast; Hoogerheide JC; Microcalorimetry, in combination with conventional methods for determining metabolic activity, opens the possibility to study the efficiency with which ATP, produced as a result of metabolic activity, is utilized by the cell for energy-requiring synthetic reactions . Using commercial baker's yeast as a test organism and glucose, ethanol, acetic and lactic acids as substrates, the fate of the ATP produced by the respiratory process was studied by measuring oxygen consumption (using the Warburg technique) and the corresponding heat development (using the LKB Flow-Microcalorimeter) . From these data heat development per mm3 oxygen consumed was calculated . Values obtained should fall within a heat production range that can be calculated from the combustion heat of the process (maximum heat development) and maximum energy conservation, assuming full participation of ATP in energy-requiring synthetic reactions (minimum heat development) . It was found that during the respiratory process of "resting" cells of baker's yeast, regardless of the substrate used, heat development was close to the maximum value inherent with substrate oxidation . Consequently, practically all ATP, produced as a result of the respiratory process, is de-phosphorylated under heat development and thus is not (or very inefficiently) utilized for energy-requiring synthetic reactions . In accordance with this conclusion it was found that addition of 2-4-DNP, a powerful uncoupler of phosphorylation from the respiratory process, did not result in an appriciable increase in heat development . Even in the presence of an assimilable N source, allowing unrestricted growth, initially only a very small percentage of the ATP produced is utilized for synthetic processes . A gradual improvement of this poor economic ATP utilization was observed during the prelogarithmic growth phase . As a possible explanation of this wasteful aerobic metabolism of baker's yeast and its restricted ability to utilize ATP for synthetic processes was mentioned the exceptional low content of messenger RNA, typical for a baker's yeast subjected to a ripening process before harvesting.

Can J Biochem, 1975 Dec, 53(12), 1316 - 22
Purification and properties of phenylalanyl-tRNA synthetase from baker's yeast; Hossain A; In an effort to avoid proteolytic fragmentation of enzymes extracted from yeast cells, the (L-phenylalanine:tRNAPhe ligase (AMP-forming) phenylalanyl-tRNA synthetase (EC6.1.1.20)) has been isolated from toluene lysates of baker's yeast in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride . The procedure includes ammonium sulfate fractionation and chromatography on DEAE-cellulose and hydroxylapatite columns . Acrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate indicates a single subunit of 75 000; other isolations have led to two subunits of 75 000 and 63 000, respectively, in agreement with other workers . Steady state kinetic analysis of the enzyme has also been carried out . The apparent kinetic patterns resulting from application of Cleland's procedure, in which the substrates are varied pairwise in the presence of saturating concentrations of the third component, suggest a reaction mechanism in which ATP and phenylalanine enter the reaction in an obligatory ordered fashion but do not completely eliminate a random mechanism.

Arch Microbiol, 1975 Nov 7, 105(3), 187 - 92
The isolation and characterization of peroxisomes (microbodies) from baker's yeast, Saccharomyces cerevisiae; Parish RW; Peroxisomes were isolated form derepressed (lactose grown) Saccharomyces cerevisiae cells following homogenization with a "Merkenschlager" cell mill (at 0 degrees C using glass beads) . Catalase and urate oxidase, along with low activities of D-amino acid oxidase and L-alpha-hydroxyacid oxidase (glycollate oxidase), were associated with the peroxisomes . No catalase activity was present in glucose repressed cells . When protoplasts prepared from derepressed cells were used for peroxisome isolation, catalase activity was not sedimentable through gradients . Apparently peroxisomes were destroyed as the cells became fermentative during protoplast preparation . The distribution of glyoxylate cycle enzymes was examined . Isocitrate lyase was not sedimentable, suggesting that, if the enzyme is peroxisome-associated, it is either readily released of present in a labile second class of peroxisomes . Low activities of malate dehydrogenase and citrate synthetase were found in peroxisome fractions from gradients, but may represent mitochondrial contamination . Citrate synthetase was not found associated with a low-density particle as had been previously reported.

Hoppe Seylers Z Physiol Chem, 1975 Nov, 356(11), 1811 - 9
{Reactivity of the 3'-terminal oligonucleotide Sequence C-A-C-C-A of tRNAPhe and tRNAVal from baker's yeast upon N-oxidation with monoperphthalic acid as compared to the oligonucleotides C-A-C-C-A and A-A-A-U-C-A-C-C-A (author's transl)}; Solfert R et al.; The nucleobases adenine and cytosine were subjected to N-oxidation by monoperphthalic acid at pH 7 and 0 degrees C in tRNAPhe, in the 3'-terminal pentanucleotide C-A-C-C-A from tRNAPhe, as well as in tRNAVal and in the 3'-terminal nonanucleotide A-A-A-U-C-A-C-C-A from tRNAVal . In the tRNAs, the oxidative attack occurs stepwise from the 3'-end . First the 3'-terminal adenine is oxidized, then the following two cytosines, then the following adenine . The oligonucleotides are far more reactive than the identical sequences in tRNA and are not oxidized according to a sequential mechanism.

Biochem J, 1975 Oct, 151(1), 67 - 73
S-adenosylmethionine decarboxylase from baker's yeast; Poso H et al.; 1 . S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'{(methylethanediylidene)dinitrilo}diguanidine) {Pegg, (1974) Biochem J . 141, 581-583} . The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4 . 2 . S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity {5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16} during the purification procedure . 3 . Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine . 4 . Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine . The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine . 5 . The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM . 6 . Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme . 7 . Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate.

Experientia, 1975 Sep 15, 31(9), 1008 - 10
Presence of a specific uridine 5'-monophosphate pyrophosphorylase in baker's yeast; Natalini P et al.; Uridine 5'-monophosphate pyrophosphorylase was found to be present in baker's yeast . The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.

Biochim Biophys Acta, 1975 Sep 8, 404(1), 142 - 51
Levels and turnover of the proteinase B inhibitors in yeast; Betz H; The ratio of the proteinase B inhibitors IB1 and IB2 from baker's yeast was shown to depend on the yeast strain by specific immunoprecipitation from boiled yeast extract and subsequent electrophoresis of the heat-dissociated precipitates on polyacrylamide gels . Bothe IB1 and IB2 were found, IB2 being by far predominant . Saccharomyces carlsbergensis NCYC 74 contained IB1, whereas in Saccharomyces cerevisiae X 2180 only IB2 was present . When cells of the latter strain were labelled with {14C} leucine from the beginning of growth and pulsed with {3H} leucine during the stationary phase, no short-lived IB1 could be detected . However, the peak of IB2 resolved on the gel showed an increased 3H/14C ratio in comparison to the majority of the other cellular proteins . The increased 3H/14C ratio was found to be the result of catabolite repression of inhibitor synthesis during exponential growth: cells growing on glucose as carbon source contain high inhibitor levels only during the stationary phase of growth, whereas during growth on acetate high amounts of inhibitor are present even in exponentially growing cells . During the stationary phase of growth the inhibitor is degraded with the same half-life as the total cellular proteins (about 50 h).

Mikrobiologiia, 1975 Sep-Oct, 44(5), 832 - 8
{Effect of nitrogen and phosphorus in the medium on the level of reserve carbohydrates in the cells of a dividing baker's yeast culture}; Bocharova NN et al.; Changes in the content of reserve carbohydrates (trehalose, glycogen) were studied in the cells of baker's yeast during their continuous cultivation at various doses of nitrogen and phosphorus in the medium . The content of reserve carbohydrates in the cells, and their viability, increased with a decrease of nitrogen in the medium . Phosphorus displayed the opposite effect, which was however insignificant within the range of its concentrations used in the experiments.

Prikl Biokhim Mikrobiol, 1975 Sep-Oct, 11(5), 662 - 8
{Effect of temperature and the active acidity of the medium on the metabolism of reserve carbohydrates and the survivability of baker's yeast}; Chernysh VG et al.; The effect of the cultivation temperature and active acidity of the medium on the contrent of trehalose and glycogen in baker's yeast was investigated . With the temperature growth from 20 to 40 degrees C the trehalose content in the cells increased, thus enhancing survivability of yeast during storage . The same results were observed upon temperature rise at the end of cultivation . pH of 4.5-5.0 intensified the synthesis of both reserve carbohydrates in baker's yeast.

Biochim Biophys Acta, 1975 Aug 13, 399(2), 375 - 83
Inhibition of thiamine transport in anaerobic baker's yeast by iodoacetate, 2,4-dinitrophenol N,N'-dicyclohexylcarbodiimide and fatty acids; Iwashima A et al.; 1 . {14C}Thiamine uptake by baker's yeast (Saccharomyces cerevisiae) was strongly inhibited by 0.2 mM iodoacetate, 0.2 mM 2,4-dinitrophenol and 0.1 mM N,N'-dicyclohexylcarbodiimide under anaerobic conditions . 2 . The inhibition of anaerobic {14C}thiamine uptake by these inhibitors was accompanied by almost parallel decreases in the ATP level of the yeast cells . 3 . On the other hand, the short-chain fatty acids inhibited {14C}thiamine uptake to a large extent, without greatly affecting the intracellular ATP level . This suggests that the acids primarily block the use of energy from ATP for the transport rather than the fermentation process . 4 . Caproate, which has a most pronounced inhibitory effect on {14C}thiamine uptake, significantly prevented the dissipation of an energized membrane state of yeast cells necessary for the active transport of thiamine . 5 . Possible ways in which the inhibitors may affect thiamine uptake were discussed.

Biochemistry, 1975 Aug 12, 14(16), 3518 - 26
The influence of amino acid substitutions on the conformational energy of cytochrome c; Warme PK; Conformational energies have been evaluated for each of the staggered side-chain conformations associated with the 261 amino acid substitutions known to occur among 60 eucaryotic species . At least 86% of these substitutions can be sterically accommodated (one at a time) within the structure of horse-heart cytochrome c resulting from conformational energy refinement . Simultaneous incorporation of all pertinent amino acid substitutions found in eight representative species into the refined horse-heart structure is also shown to be sterically possible, with few exceptions . In two cases (Pekin duck cytochrome with 10 substitutions and Samia cynthia cytochrome with 24 substitutions), all substitutions could be readily incorporated, and the total energies associated with their computed structures differed by less than 10 kcal/mol from that of horse-heart cytochrome c . In the cytochromes from rattlesnake (22 substitutions), tuna (18 substitutions), and Neurospora crassa (36 substitutions), tyrosine could not be substituted for phenylalanine at position 46, within the constraints of the calculations . However, when all of the remaining substitutions were incorporated into these three cytochromes, their computed conformational energies differed by less than 30 kcal/mol from that of horse-heart cytochrome c . Between two and four amino acid substitutions cause high energies in the cytochromes from human, baker's yeast, and cotton seed, but all of the remaining substitutions are consistent with a low energy conformation . These results suggest that the structures of homologous proteins may be even more similar than has previously been recognized . Substitutions of all possible amino acid types at the invariant positions (where all eucaryotic cytochromes c bear the same amino acid) have revealed some cases where different amino acids can be accommodated, thus demonstrating that the biological constraints on amino acid substitutions are often different from the purely steric constraints investigated in this work.

Biochemistry, 1975 Jul 29, 14(15), 3310 - 4
The binding of complementary oligoribonucleotides to yeast initiator Transfer RNA; Freier SM et al.; Oligoribonucleotide binding to baker's yeast initiator tRNA was measured by equilibrium dialysis in order to determine which regions of the tRNA were free to bind complementary oligomers and which were involved in secondary and tertiary structure . Association constants of trinucleoside diphosphates and tetranucleoside triphophates complementary to the single-stranded regions of the cloverleaf structure of yeast tRNAfMet were measured at o degrees in 1.0 M NaCl, and 0.01 M MgCl2 . The only regions of the tRNA whose complementary oligomers bound to the tRNA were the amino acid acceptor end and the five nucleotides at the 5' end of the anticodon loop . These results differ from those for the other tRNAs studied by this technique; usually oligomers complementary to the dihydrouracil loop bind to the tRNA . The sequence of yeast tRNAfMet and other eucaryotic initiators is unusual . The "TpsiC loop" contains the sequence A-U-C instead of T-psi-C, yet the binding pattern to the THE TpsiC LOOP IS LIKE THAT FOR OTHER TRNAs; no oligomers bind.

Biochim Biophys Acta, 1975 Jul 27, 397(1), 117 - 23
Protamine kinase from yeast; Nakashima M et al.; A protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) which preferentially phosphorylates protamine is purified about 250-fold from the soluble fraction of baker's yeast (Saccharomyces cerevisiae) . This enzyme is not sensitive to activation by cyclic nucleotides . Histone is about 5% as active as protamine in the reaction rate . Neither casein, phosvitin nor glycogen phosphorylase is active as substrate . The enzyme is distinguishable from casein kinase of the classical type (Rabinowitz, M . and Lipmann, F . (1960) J . Biol . Chem . 235, 1043-1050) and from adenoshine 3', 5'-monophosphate-dependent protein kinase described earlier (Takai, Y., Yamamura, H . and Nishizuka, Y . (1974) J . Biol . Chem . 249,530-535).

Biochemistry, 1975 Jul 15, 14(14), 3278 - 91
Proton magnetic resonance studies on the conformation of the hexanucleotide, GmpApApYpApsiP, and Related fragments from the anticodong loop of baker's yeast phenylalanine transfer ribonucleic acid; Kan LS et al.; A hexanucleotide, GmpApApYpAppsiP, and 11 related compounds from the anticodon loop of Baker's yeast tRNA-Phe were studied by proton magnetic resonance from 100 to 250 MHz . Totally 19 resonance lines from all the base protons (H8, H6, and H2), H1', and methyl proton resonances of the hexamer have been assigned by a systematic "incremental procedure" in comparing all the related shorter fragments . Emphasis is given to the Y base and its stacking conformation with respect to its nearest neighboring bases . The results reveal a strong tendency of the purine bases to have a maximal extent of base-base overlap with their neighbors in the sequence . This tendency is manifested in a zigzag (or balcony-like) mode of base-stacking pattern of the -ApYpA-sequence in the hexamer in which the -pA-residue tends to stack toward the adduct ring (C10, C11, and N12) of Y . This tendency is also shown in the formation of a stack of GmAAA closing the gap left behind by the excision of Y in the hexamer GmpApAp--pApp . The implication of these findings to the structure and function of tRNA is discussed.

Prikl Biokhim Mikrobiol, 1975 Jul-Aug, 11(4), 500 - 5
{Optimization of cultivation conditions of baker's yeast}; Palagina NK et al.; By an integrated factorial experiment optimal proportions of the quantity of inoculation yeast, molasses dosage and degree of their initial water dilution have been established for the periodic yeast cultivation . An equation describing the process has been derived . This equation makes is possible to assess yeast yield at any value of the above parameters within their extreme limits . The yeast cultivation under optimized conditions results in a 25% increase of the economic coefficient coefficient of reproduction as compared with the control, thus approximating the theoretically conceivable value.

Can J Biochem, 1975 Jul, 53(7), 735 - 46
Analysis of O2'-methylnucleoside 5'-phosphates in snake venom hydrolysates of RNA: identification of O2'-methyl-5-carboxymethyluridine as a constituent of yeast transfer RNA; Gray MW; Snake venom phosphodiesterase liberates the O2-methylnucleoside (Nm) constituents of RNA as the corresponding 5-nucleotides (PNm), which, in contrast to normal 5-nucleotides (pN), are resistant to dephosphorylation by venom 5-nucleotidase . This property provides the basis of a convenient and highly reproducible quantitative assay for Nm residues in RNA . The assay method involves: (1) hydrolysis of RNA with whole or partially-purified snake venom; (2) isolation of the pNm derivatives, as a group, by anion-exchange chromatography on DEAE-cellulose; (3) resolution of the individual pNm compounds by two-dimensional paper chromatography; (4) identification and quantitative measurement of pNm derivatives by ultraviolet absorption spectrophotometry . Using this procedure, the molar proportions of the Nm constituents of wheat embryo, yeast, and Escherichia coli tRNA have been determined . The close correspondence between the values measured by venom hydrolysis and those obtained by analysis of alkali-stable dinucleotide (Nm-Np) sequences attests to the validity of the venom assay, and further indicates that alkali-stable sequences larger than dinucleotides are not present in significant amounts in the tRNA of the above three organisms . During the present investigation, several ultraviolet-absorbing components, not immediately identifiable as ribose-methylated nucleotides, were isolated along with the expected O2-methylnucleoside 5-phosphates . Preliminary characterization of one of these compounds suggests that it is a derivative of a novel nucleoside, O2-methyl-5-carboxymethyluridine (cm5Um is released as part of an alkali-stable dinucleotide, cm5Um-Ap . The proportion of pU-2 in venom hydrolysates of yeast tRNA (0.02 mol percent, the same as the content of cm5Um-Ap in alkaline hydrolysates) suggests that O2-methyl-5-carboxymethyluridine may be confined to a single isoaccepting species of tRNA in yeast . In an allied study, reinvestigation of the alkali-stable dinucleotide sequences of baker's yeast tRNA has confirmed previous results concerning the sequence distribution of O2-methylribose in yeast tRNA (Gray, M . W . & Lane, B.G . (1967) Biochim . Biophys . Acta 134, 243-257).

J Bacteriol, 1975 Jun, 122(3), 1265 - 73
Three yeast proteins that specifically inhibit yeast proteases A, B, and C; Lenney JF; Baker's yeast was found to contain inhibitors of yeast proteases A and C . These two proteins were partially purified, characterized, and compared with the previously described inhibitor of protease B . The A and B inhibitors were very thermostable and were extracted from intact yeast cells at 9k C . The A inhibitor appeared to be a protein with a molecular weight of about 22,000 which could be dissociated into two monomers or chains, both of which had a molecular weight of approximately 11,000 . The protease C (carboxypeptidase Y)-inhibitor complex was purified and then partially disociated on an ion-exchange column . The free protease C inhibitor was very unstable, possibly because of destruction by a contaminating protease . Each inhibitor was specific for its corresponding protease and each inhibition was competitive . Whereas proteases A, B, and C destroyed the B inhibitor, only protease B had a pronounced destructive effect on the protease A inhibitor . Pepstatin was found to be a selective inhibitor of protease A, whereas chymostatin and antipain specifically inhibited protease B.

Biochim Biophys Acta, 1975 May 23, 391(1), 67 - 74
Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast; Mildner P et al.; In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions . Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts . Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose . The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP . This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites . The two activities differ in thermostability and only one activity could be completely abolished by heat treatment . The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP . ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis . Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.

Biochemistry, 1975 May 6, 14(9), 2009 - 14
Amino acid sequence of two functional sites in yeast glycogen phosphorylase; Lerch K et al.; The structure of two functional sites in baker's yeast (Saccharomyces cerevisiae) glycogen phosphorylase (EC 2.4 1.1) was determined as part of a study on the evolution of regulatory enzymes . S-Carboxymethylated, MaBH4-reduced 32-P-labeled yeast phosphorylase a was cleaved with CNBr, thermolysin, and pepsin . Peptides labeled with 32-P or carrying the fluorescent pyridoxyl marker were isolated and purified using ion-exchange chromatography and gel filtration . CNBr cleavage yielded a single radioactive phosphopeptide (42 residues long) and one small fluorescent peptide with the unique sequence epsilon-Pxy-Lys-Phe-Val-Met . Thermolysin digestion gave rise to one radioactive octapeptide and two fluorescent peptides, 15 and 2 residues long, respectively . From a combination of substractive Edman degradations and digestion with yeast protease C, the sequence of the 32-P-labeled octapeptide was established . Phosphothreonine was identified as the sole phosphorylated amino acid, giving the following structure for the site involved in the covalent regulation of yeast phosphorylase: Leu-Thr(P) -Gly-Phe-Leu-Pro-Gln-Glu . The two fluorescent thermolytic peptides, together with two additional pyridoxyl peptides isolated after peptic digestion of the enzyme yielded the following sequence around the site binding pyridoxal-5'-P, the cofactor essential for phosphorylase activity: Ile-Ser-Thr-Ala-Gly-Thr-Glu-Ala-Ser-Gly-Thr-Ser-Asn-Met-Lys(P Pxy)-Phe-Val-Met . While the phosphorylated site bears no resemblance to the site of covalent control in vertebrate phosphorylases, the pyridoxal-P binding site in the yeast enayme displays remarkable homologies with its animal counterparts; the finding that 14 out of 18 amino acids are identical strongly suggests that the cofactor must be directly involved in catalysis.

Biochemistry, 1975 May 6, 14(9), 2015 - 23
Purification and properties of a yeast protein kinase; Lerch K et al.; A protein phosphokinase (EC 2.7.1.1.37) was isolated from baker's yeast (Saccharomyces cerevisiae) after a 17,000-fold purification; the purified enzyme is homogeneous according to the criteria of gel electrophoresis and ultracentrifuge analysis . The enzyme has a high isoelectric point of ca . 9 and appears to exist as a monomer with a molecular weight of 42,000 plus or minus 1500 . It is neither stimulated by cyclic 3',5'-AMP, -GMP, -CMP or -ump nor inhibited by the regulatory subunit of rabbit muscle protein kinase (Reimann, E . M., Walsh, D . A., and Krebs, E . G . (1971), J . Biol . Chem . 246, 1986) . In the presence of divalent metal ions, preferably Mg-2+ or Mn-2+, the enzyme readily transfers the terminal phosphate group of ATP to phosvitin, alphaS1B- and beta a-casein and an NH2-terminal tryptic peptide derived from beta a-casein, but not to protamine, lysine, or arginine-rich histones or to yeast enzymes such as phosphorylase, phosphofructokinase, or pyruvate carboxylase; serine and polyserine were also inactive as phosphate acceptors . Km values of 0.17 mM for beta a-casein and 0.2 mMfor ATP were determined at 10 mM Mg-2+ . The urified yeast protein kinase also catalyzes the reverse reaction, namely, the transfer of phosphate from fully phosphorylated beta a-casein or its NH2-terminal peptide to ADP resulting in the formation of ATP . AMP, GDP, UDP, and CDP did not serve as phosphate acceptors in this reaction . As observed by Rabinowitz and Lipmann (Rabinowitz, M., and Lipmann, F . (1960), J . Biol . Chem . 235, 1043) both reactions have different pHoptima with values of 7.5 for the forward reaction (phosphorylation of the proteins) and ca 5.2 for the formation of ATP; both are differently affected by salts . Phosphorylation of beta a-casein with {gamma-32-P}ATP followed by digestion of the labeled protein with trypsin indicated that all the radioactivity was exclusively introduced in an NH2-terminal peptide possessing the unique sequence: Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu...(Ribadeau-Dumas, B., Brignon, G., Grosclaude, F., and Mercier, J.-C . (1971), eur J . Biochem . 20, 264) . By subjecting beta a-casein and its NH2-terminal peptide to the combined action of almond acid phosphatease and purified yeast protein kinase, it was determined that the phosphorylation and dephosphorylation reactions proceed randomly, i.e., all seryl phosphate residues are equally susceptible and that the rate of phosphorylation decreases drastically as the number of bound phosphate groups in the substrate diminishes.

Eur J Biochem, 1975 May 6, 53(2), 487 - 92
Modification of phenylalanyl-tRNA synthetase from baker's yeast by proteolytic cleavage and properties of the trypsin-modified enzyme; Fasiolo F et al.; Earlier studies have shown that native phenylalanyl-tRNA synthetase from baker's yeast contains two different kinds of subunits, alpha of molecular weight 73000 and beta of molecular weight 63000 . The enzyme is an asymmetric tetramer alpha-2beta-2, which binds two moles of each ligand per mole . Incubation of the purified enzyme with trypsin results in an irreversible conversion: the alpha-subunit remains apparently unchanged but beta is rapidly degraded and yields a lighter species beta of molecular weight 41000 . The trypsin-modified enzyme is an alpha-2beta-2 molecule which can still activate phenylalanine but cannot transfer it to tRNA-Phe; furthermore it does not bind tRNA-Phe but its kinetic parameters are identical to those of the native enzyme with respect to ATP and phenylalanine . Therefore the two beta subunits play a critical part in tRNA binding . Isolated alpha or beta subunits exhibit no significant activity and both types of subunit seem to be required for phenylalanine activation.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1378 - 82
Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species; Von Der Haar F et al.; TRNA-Phe species from baker's yeast modified at the 3'-terminus in many cases are phenylalanylatable substrates . Out of several tRNA-Phe species possessing a modified 3'-end that cannot be phenylalanylated, only two, tRNA-Phe-C-C-2'dA and the tRNA-Phe-C-C-formycin-oxi-red, are strong competitive inhibitors for tRNA-Phe-C-C-A during phenylalanylation . In the ATP/PPi exchange, both these inhibitors reduce Vmax to about 25%; but whereas tRNA-Phe-C-C-2dA has no influence on KmATP and Km Phe during ATP/PPi exchange, tRNA-Phe-C-C-formycin-oxi-red reduces KmATP from 1430 muM, found in the absence of tRNA-Phe, to 230 muM, and Km-Phe, from 38 to 14 muM . The values found in the presence of tRNA-Phe-C-C-formycin-oxi-red during ATP/PPi exchange are identical with those determined in the phenylalanylation of tRNA-Phe-C-C-A . All other tRNA-Phe species carrying a modified 3'end that cannot be phenylalanylated exhibit a mixed competitive-noncompetitive inhibition in the phenylalanylation reaction . In the ATP/PPi exchange, they do not influence KmATP and KmPHE and only weakly, if at all, Vmax . The results show that the 3'adenosine of tRNA-Phe cannot solely be a passive acceptor for phenylalanine, but must in addition play an active role during enzyme-substrate interaction . The data can be consistently explained by the hypothesis that the 3'-adenosine of tRNA-Phe triggers a conformational change of the enzyme.

Biochim Biophys Acta, 1975 Mar 10, 383(2), 160 - 7
Purification of an inhibitor of DNA photolyase with fluorescent spectra similar to those of the enzyme; Madden JJ et al.; 1 . The binding of DNA photolyase, the enzyme which uncouples cyclobutadipyrimidines in DNA upon illumination, to its substrate was enhanced by a substance isolated from acidified autolysates of baker's yeast . 2 . This substance, referred to as activator, was partially purified by chloroform extraction, ion-exchange chromatography on Dowex 50 and DEAE-cellulose, and gel filtration on Sephadex G-10 . Upon lyophilization and storage at -20 degrees C, it was converted to a potent inhibitor of enzyme binding to substrate . 3 . The inhibitor appeared as a single band in two thin-layer chromatography systems and was detected by ultraviolet absorbance, fluorescence, and ninhydrin staining . 4 . The similarity between the fluorescence spectra of the inhibitor and the enzyme suggested that the inhibitor was structurally analogous to a chromophore in the enzyme and that the activator from which the inhibitor was derived may be the active chromophore of the enzyme.

Eur J Biochem, 1975 Mar 3, 52(1), 1 - 7
On the activity and regulation of anaplerotic and gluconeogenetic enzymes during the growth process of baker's yeast . The biphasic growth; Haarasilta S et al.; The anaplerotic and gluconeogenetic metabolism of baker's yeast was studied at the enzymatic level during glucose-ethanol diauxic growth in the presence and absence of aspartate . Of the two possible anaplerotic systems, only the pyruvate carboxylase by-pass was present during the whole growth process . The second system, the glyoxylate by-pass (isocitrate lyase as the indicator), like the specific enzymes of the gluconeogenetic metabolism, phosphoenolpyruvate carboxykinase and hexosediphosphatase began to appear only after the glucose had been consumed . The addition of glucose during the growth phase based on ethanol effected a rapid disappearance of phosphoenolpyruvate carboxykinase and hexosediphosphatase activities . The activity of pyruvate carboxylase decreased when the growth medium was supplied with asparate . The presence of aspartate had no effect on the activities of the other enzymes studied.

Mol Cell Biochem, 1975 Feb 28, 6(2), 93 - 100
Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions; Machado A et al.; The activities of isocitrate dehydrogenase (NAD), isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase have been investigated in Saccharomyces cerevisiae grown in a variety of aerobic and hypoxic conditions, the latter including oxygen deprivation, high glucose concentration, addition of inhibitors of mitochondrial protein synthesis, respiratory inhibition by azide, and impaired respiration mutants . All hypoxic conditions led to a marked decrease of oxoglutarate dehydrogenase and significant decreases of the two isocitrate dehydrogenases . According to its kinetic properties, the NAD-isocitrate dehydrogenase will not be operative in hypoxia "in vivo" . From these and other related facts it is concluded that hypoxic conditions in yeast generally lead to a splitting of the tricarboxylic acid cycle and that glutamate synthesis in these conditions takes place through the coupling of the NADP-linked isocitrate and glutamate dehydrogenases.

Biochim Biophys Acta, 1975 Feb 19, 377(2), 331 - 42
Biosynthesis of acid phosphatase of baker's yeast . Characterization of a protoplast-bound fraction containing precursors of the exo-enzyme; Boer P et al.; 1 . Yeast protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M., Boer, P . and Steyn-Parve, E.P . (1972) Biochim . Biophys . Acta 268, 431-441) . The major part (70%) of this protoplast-bound acid phosphatase can be solubilized by nonionic detergents, such as Triton X-100 . 2 . The kinetics of radioactive amino acid incorporation in the solubilized and in the secreted enzyme has been estimated by pulse-chase labelling of secreting protoplasts, followed by fractionation and counting radioactivity in the enzyme band in polyacrylamide gels after electrophoresis at pH 5.0 . A precursor-product relationship between the Triton X-100-extractable fraction of the protoplast-bound acid phosphatase and the secreted enzyme is apparent . 3 . The solubilized acid phosphatase is essentially indistinguishable from the secreted enzyme with regard to a number of enzymatic properties and its stability towards pH and temperature . Both enzymes also behave alike on polyacrylamide-gel electrophoresis, producing a single acid phosphatase band with glycoprotein character and comparable mobility . 4 . A striking difference is seen in isopycnic equilibrium sedimentation in CsCl: the secreted acid phosphatase is homogeneous, with a buoyant density of p equals 1.47 g/cm3, while the Triton X-100-extractable part of the protoplast-bound acid phosphatase is heterogeneous; besides heavier material a major component with buoyant density of p equals 1.37 g/cm3 is always visible.

Chem Phys Lipids, 1975 Feb, 14(1), 15 - 32
Neutral lipids in the cells and cell envelope fractions of aerobic baker's yeast and anaerobic brewer's yeast; Nurminen T et al.; The neutral lipids from whole cells and cell envelopes of aerobic Saccharomyces cerevisiae and anaerobic Sacch . carlsbergensis and the cell walls isolated from the cell envelopes were analysed . The effect of anaerobiosis was particularly clear on the neutral lipid composition of the plasma membrane . Compared to the anaerobic membrane, the aerobic membrane contained more C16:1, C18:1 and other unsaturated fatty acids, more total sterol, more than ten times as much ergosterol and less than one tenth as much squalene, reflecting differences between the aerobic and anaerobic whole cellmthe main sterol in the aerobic membrane ergosterol, was mainly in the free form, whereas zymosterol, 24(28)-dehydroergosterol, epi- or fecosterol and lanosterol were predominantly esterified . In contrast, the anerobic membrane contained small amounts of biosynthetic sterol precursors of ergosterol (mainly esterified), and was clearly richer in saturated fatty acids having a greater variation in chain length and in 18:2 acid . Both plasma membranes contained a considerable amount of triacyglycerols, while the amount of lower acylglycerols was clearly higher in the anaerobic plasma membrane . The lipid composition of both cell walls were relatively similar, consisting mainly of triacylglycerols and lower acylglycerols.

J Biol Chem, 1975 Jan 25, 250(2), 767 - 74
Cytochrome c oxidase from bakers' yeast . V . Arrangement of the subunits in the isolated and membrane-bound enzyme; Eytan GD et al.; Cytochrome c oxidase from baker's yeast contains three mitochondrially made subunits (I to III) which are relatively hydrophobic and four cytoplasmically made subunits (IV to VII) which are relatively hydrophilic (Mason, T . L., Poyton, R . O., Wharton, D.C., and Schatz, G . (1973) J . Biol . Chem . 248, 1346-1354 and Poyton, R . O., and Schatz, G . (1975) J . Biol . Chem . 250, 752-761) . In order to explore the arrangement of these subunits in the holoenzyme, the reactivity of each subunit with a variety of "surface probes" was tested with isolated cytochrome c oxidase, with cytochrome c oxidase incorporated into liposomes, and with mitochondrially bound cytochrome c oxidase . The surface probes included iodination with lactoperoxidase and coupling with p-diazonium benzenesulfonate . In addition, external subunits were identified by linking them to bovine serum albumin carrying a covalently bound isocyanate group . In the membrane-bound enzyme, Subunit I was almost completely inaccessible and Subunit II was partly inaccessible to all surface probes . All of the other subunits were accessible . Similar results were obtained with the solubilized enzyme, except that the differences in reactivity between the individual subunits were less clear-cut . The results obtained with liposome-bound cytochrome c oxidase resembled those obtained with the mitochondrially bound enzyme . These data suggest that the two largest mitochondrially made subunits are localized in the interior of the enzyme and that they are genuine components of cytochrome c oxidase.

C R Acad Sci Hebd Seances Acad Sci D, 1975 Jan 20, 280(3), 315 - 7
{Effect of acetaldehyde upon the oxidative degradation of glucose by baker's yeast}; Iscaki M et al.; Acetaldehyde and glucose are simultaneously oxided by baker's yeast . By the use of 14C-U-glucose, as well as 14C-1 or 14C-6 glucose, it has been possible to demonstrate that glucose oxidation can partially be substituted by acetaldehyde oxidation . Acetaldehyde increases the 14C-1/14C-6 ratio of the evolved carbon-dioxide, thus stimulating the amount of glucose degraded by the phosphogluconate route and decreasing the amount metabolised by the glycolytic pathway.

Mol Biol (Mosk), 1975 Jan-Feb, 9(1), 55 - 62
{The primary structure of tRNA Val 2a from baker's yeast}; Aksel'rod VD et al.; The primary structure of tRNAVal2a from baker's yeast has been determined . The general methods of the investigation are presented . Twenty six distinguished points can be noted in the tRNAVal2a and tRNA1Val from baker's yeast . The anticodon region of tRNAVal2a is represented by the sequence NAC, where N corresponds to a uridine analogue nucleoside of unknown structure . The comparison of primary structures of tRNAVal2a, tRNAVal2a, tRNA1Val from E . coli and tRNAVal2a and tRNA1Val from baker's yeast is analysed.

Nahrung, 1975, 19(9-10), 885 - 90
Biological value of the biomass and protein isolates from different yeasts; Charatjan SG et al.; The microbiological analysis with the test-organism Tetrahymena pyriformis W can be used successfully to measure the relative nutritive value of the biomass and protein isolates from different yeasts for screening the groups of micro-organisms to choose those of the desired quality . In general, this method is rapid, comparatively cheap and gives the possibility to test large numbers of samples simultaneously . According to the literature and our own results, the relative nutritive value, the percentage of absorbed "N", retained by test-organism and used for its growth and multiplication expressed as cell count and given in relation to a standard protein (casein) is generally in a good agreement with results obtained with animals . This microbiological method is promising for investigating the influence of certain technological operations on the quality of protein . The investigation has been performed by the use of Baker's yeast Saccharomyces cerevisiae, of fodder yeast Candida, grown on oil hydrocarbons and on wood hydrolysates, yeasts Hansenula and Mycoderma, grown on a medium containing ethanol as the sole carbon source . The best results were obtained with the yeast grown on the ethanol-containing medium . The biological value of the biomass of yeast Hansenula is 91% and of protein isolate 96% as related to casein as the standard protein.

Acta Chem Scand B, 1975, 29(9), 932 - 6
Putrescine-insensitive S-adenosyl-L-methionine decarboxylase from Tetrahymena pyriformis; Poso H et al.; Extracts of Tetrahymena pyriformis contain a soluble S-adenosyl-L-methionine decarboxylase which, in contrast to the enzyme from most eukaryotic organisms, is not stimulated by putrescine or spermidine . The protozoan adenosylmethionine decarboxylase, unlike the putrescine-insensitive enzyme form Escherichia coli, did not require any metal ions for catalytic activity either . Adenosylmethionine decarboxylase from Tetrahymena resembled the prokaryotic enzyme as far the inhibition by methylglyoxal bis(guanylhydrazone) was concerned, but behaved more like putrescine-activated enzyme in regard to the inhibition by 4-bromo-3-hydroxy benzyl-oxyamine . Adesylmethionine decarboxylase from rat liver, baker's yeast, E . coli and Tetrahymena were strongly inhibited by S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine), the product of the reaction . The function of adenosylmethionine decarboxylase from Tetrahymena like that of the enzymes from other organisms appears to be closely connected to the synthesis of spermidine.

Radiat Environ Biophys, 1975, 11(4), 295 - 307
Studies on the energy metabolism during anaerobic fermentation of glucose by baker's yeast; Hoogerheide JC; As a result of the intimate association of ADP phosphorylation with alcoholic fermentation, resulting in the synthesis of 2 mole ATP per mole glucose fermented, it may be calculated that a minimum of 672 mucal heat development may be expected for every mm-3 CO2 developed during alcoholic fermentation . When all ATP produced would be fully de-phosphorylated to ADP + Pi (e.g . by ATP-ase activity) a maximum heat development of 1200 mucal per mm-3 CO2 could be expected . Using the LKB-Flow-Microcalorimeter for measurement of heat development and at the same time the Warburg technique for measuring CO2 development during anaerobic glucose fermentation of a baker's yeast suspension, the heat development per mm-3 CO2 produced was calculated over a fermentation period of 90 min . Maintenance of strict anaerobic conditions in the Flow-Microcalorimeter vessel was complicated by diffusion of traces of oxygen via the Teflon transport lines, resulting in excessive heat development values, not representative for the alcoholic fermentation . This problem could be circumvented by removal of traces of oxygen by means of addition of the enzyme glucose-oxidase . Poisoning the respiratory enzyme system of the yeast by addition of KCN or azide, or using respiratory-deficient mutants of the yeast also resulted in heat development values, inherent with alcoholic fermentation . The values obtained were very close to the minimum of 672 mucal per mm-3 CO2, at least during the initial phases of fermentation, indicating that ADP regeneration from ATP, essential for maintaining the high fermentation rate, is not primarily the result of ATP-ase activity, but must be due to participation of ATP in energy-requiring synthetic reactions.

Physiol Chem Phys, 1975, 7(6), 555 - 64
The structural subunit of glucose-6-phosphate dehydrogenase (baker's yeast); Robbins JE et al.; The subunit molecular weight of glucose-6-phosphate dehydrogenase (G6PD) from baker's yeast has been evaluated . The subunit molecular weight value is shown to be 25,500 daltons by analytical ultracentrifugation, SDS-polyacrylamide gel electrophoresis, and the number of peptides produced by CNBr cleavage . The number of NADP binding sites was determined to be one per 25,500 dalton unit.

Folia Microbiol (Praha), 1975, 20(6), 496 - 503
Transport of 4-deoxy- and 6-deoxy-D-glucose in baker's yeast; Kotyk A et al.; Tritium-labelled 4-deoxy-D-glucose (4-dglc) and 6-deoxy-D-glucose (6-dgcl) were prepared by catalytic hydrogenolysis of the corresponding deoxyiodo derivatives with gaseous tritium . The two sugars are transported into Saccharomyces cerevisiae by both the constitutive glucose and the inducible galactose carrier . Uranyl ions are powerful inhibitors . The pH optimum in uninduced cells lies at 5.5 for both sugars, the apparent activation energies (between 15 and 35 degrees C) are 25.1 kJ/mol and 16.5 kJ/mol, respectively . The steady-state intracellular concentration of both sugars is less than the extracellular one (no uphill transport) . Neither of them is a substrate of yeast hexokinase . 4-Deoxy-D-glucose undergoes a dinitrophenol-sensitive conversion to an unknown metabolite which is not phosphorylated and may represent one of its oxidation products.

J Lipid Res, 1973 Jul, 14(4), 406 - 14
2,3-Erythro-dihydroxyhexacosanoic acid and homologs: isolation from yeast cerebrin phosphate and determination of their structures; Hoshi M et al.; Homologs of methyl esters of very polar fatty acids were obtained by methanolysis of cerebrin phosphate isolated from baker's yeast . The major ester component was isolated by preparative gas-liquid chromatography and was found to be 2,3-dihydroxyhexacosanoic acid as deduced from the mass spectra of its trimethylsilyl ether and isopropylidene derivative, reaction with periodate, and comparison of its chromatographic behavior with that of synthetic erythro- and threo-dihydroxy acids . Its infrared spectrum supported the above conclusions . From their retention times by gas-liquid chromatography, the homologs were found to be saturated, unbranched 2,3-dihydroxy fatty acids with 24-27 carbon atoms . The synthesis of the new fatty acids, erythro- and threo-2,3-dihydroxyhexacosanoic acids, is also reported . A method for separating trans-2-hexacosenoic acid, a key intermediate of the above synthesis, and its isomer, trans-3-hexacosenoic acid, both formed by dehydrobromination of 2-bromohexacosanoic acid, is also described.

J Lipid Res, 1967 May, 8(3), 281 - 2
Removal of the sphingolipid impurity from preparations of yeast phosphatidyl inositol; Trevelyan WE; A complex sphingolipid containing inositol and mannose, present in lipid extracted from toluene-autolyzed baker's yeast, was eluted from silicic acid columns immediately after phosphatidyl inositol, and was the main nitrogenous impurity in crude preparations of this phospholipid . Nitrogenfree phosphatidyl inositol was obtained by rechromatography on alumina . Modifications to the chromatographic procedure also gave diphosphatidyl glycerol containing the theoretical 4.29% P.

J Lipid Res, 1966 May, 7(3), 445 - 7
Preparation of phosphatidyl inositol from baker's yeast; Trevelyan WE; Phosphatidyl inositol and a cardiolipin-like phospholipid survive the autolysis which is induced in yeast by treating it with toluene, whereas other phospholipids are extensively degraded . Lipid from autolyzed yeast was suspended in isopropanol to extract lecithin and neutral lipid . The insoluble phosphatidyl inositol and cardiolipin were then separated by chromatography on silicic acid.

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