Yeast Preparations Research Articles

The absorption of protons with specific amino acids and carbohydrates by yeast.

1. Proton uptake in the presence of various amino acids was studied in washed yeast suspensions containing deoxyglucose and antimycin to inhibit energy metabolism. A series of mutant strains of Saccharomyces cerevisiae with defective amino acid permeases was used. The fast absorption of glycine, l-citrulline and l-methionine through the general amino acid permease was associated with the uptake of about 2 extra equivalents of protons per mol of amino acid absorbed, whereas the slower absorption of l-methionine, l-proline and, possibly, l-arginine through their specific permeases was associated with about 1 proton equivalent. l-Canavanine and l-lysine were also absorbed with 1-2 equivalents of protons. 2. A strain of Saccharomyces carlsbergensis behaved similarly with these amino acids. 3. Preparations of the latter yeast grown with maltose subsequently absorbed it with 2-3 equivalents of protons. The accelerated rate of proton uptake increased up to a maximum value with the maltose concentration (K(m)=1.6mm). The uptake of protons was also faster in the presence of alpha-methylglucoside and sucrose, but not in the presence of glucose, galactose or 2-deoxyglucose. All of these compounds except the last could cause acid formation. The uptake of protons induced by maltose, alpha-methylglucoside and sucrose was not observed when the yeast was grown with glucose, although acid was then formed both from sucrose and glucose. 4. A strain of Saccharomyces fragilis that both fermented and formed acid from lactose absorbed extra protons in the presence of lactose. 5. The observations show that protons were co-substrates in the systems transporting the amino acids and certain of the carbohydrates.

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis.

1. [(14)C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO(3) (-). In a cell-free yeast preparation addition of HCO(3) (-) stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of beta-hydroxy-beta-methylglutaryl-CoA formed from [2-(14)C]- and [1,3-(14)C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-(14)C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-(14)C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.

Lactase Potential of Various Microorganisms Grown in Whey

Microbial cell preparations from selected microorganisms were evaluated for their ability to hydrolyze lactose to monosugars in a pH adjusted whey medium. Optimal pH and temperature for such activity were established.Freeze-dried crude cell preparations of lactase from mold, yeast, and bacteria, depending on species and strain, hydrolyzed 5 to 80% lactose in nutrient whey during 5-hour incubation under optimal conditions. Molds had the lowest lactase activity but the highest cell yields, and bacteria produced the highest lactase activity and lowest cell yields. Activity was increased up to 34% with freeze-dried yeast and bacteria cell preparations given individual physical and chemical treatments. Washing with toulene proved most effective. Mold preparations gave lower activity after similar treatments.Crude cell preparations showing highest lactase activity in deproteinized whey medium among molds were Neurospora crassa; the yeast, Saccharomyces fragilis; and the bacterium, Lactobacillus hel-veticus.

Viability of Commercial “Household” Yeast Preparations

Viability of Commercial “Household” Yeast Preparations

The effect of a complex yeast preparation, as a food supplement, on the growth of Ehrlich's ascites tumour in mice

The effect of a complex yeast preparation, as a food supplement, has been investigated on the growth of Ehrlich's ascites tumour in ascitic and solid form in mice. Animals were premedicated with the preparation for 14 days before cancer inoculation, and medication then continued to completion of the experiment. Tumour growth was reduced in all groups treated with the preparation, as compared to control animals treated with saline. The preparation was more effective in male animals than in female animals. The preparation appears to antagonise the establishment (“take”) and initial growth of the cancer. Its possible mechanism of action is discussed.

The mechanism of S-adenosyl-L-methionine synthesis by purified preparations of bakers' yeast.

The mechanism of S-adenosyl-L-methionine synthesis by purified preparations of bakers' yeast.

The readily extracted lipids of Histoplasma capsulatum and Blastomyces dermatitidis

1. 1. Cell walls and cell sap (lyophilized preparation of whole cell minus cell wall) of both yeast and mycelial phases of Histoplasma capsulatum and Blastomyces dermatitidis were extracted with chloroform-methanol and the lipids thus derived were compared. 2. 2. There was little difference between the total readily extracted lipid of the cell sap preparations of yeast and mycelial phases of H. capsulatum. Cell saps of B. dermatitidis generally contained less lipid than H. capsulatum; the mycelial phase contained about half that of the corresponding yeast phase preparations. Most cell walls had less than 3% lipid, with less m mycelial phase than corresponding yeast phase walls. 3. 3. Cell wall and cell sap extracts regardless of source contained phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, triglycerides, diglycerides, sterols, sterol esters and free fatty acids. Triglycerides were the greatest single component in the extracts; phosphatidyl choline and phosphatidyl ethanolamine were the most concentrated phospholipids. Quantitative densitometry of these three classes of compounds revealed little difference between cell wall and cell sap preparations, or yeast and mycelial phase preparations, that could not be explained on the basis of incubation time or different rates of metabolism in different media. 4. 4. The major differences between cell walls and homologous cell sap, and yeast phase and mycelial phase, involved primarily the quantities of oleic and linoleic acids in the extracts. These two fatty acids together, however, accounted for 75–80% of the total fatty acids of all extracts; the relative proportions of the two distinguished the preparations. In general, oleic acid predominated in yeast phase extracts; linoleic acid was either equal or greater in concentration in the mycelial phase extracts. Cell walls usually contained more oleic acid and less linoleic acid than homologous cell saps. This was particularly obvious when the fatty acids of the lipid classes were examined. Even though differences were demonstrated between cell wall and cell sap preparations, and yeast and mycelial phase preparations, little difference could be demonstrated between analogous lipid extracts of H. capsulatum and B. dennatitidis.

Stoicheiometrical proton and potassium ion movements accompanying the absorption of amino acids by the yeast Saccharomyces carlsbergensis.

1. Proton uptake into the yeast Saccharomyces carlsbergensis, was studied at pH4.5-5.5 in the presence of both antimycin and 2-deoxyglucose to inhibit energy metabolism. Previous work had shown that the cells then absorbed about 20nmol of glycine or l-phenylalanine against a considerable amino acid concentration gradient. The addition of the amino acid immediately stimulated the rate of uptake of protons two- to three-fold. About 2 extra equivalents of H(+) accompanied a given amount of the amino acids into the yeast preparations exposed to the metabolic inhibitors for 2-4min and about 1.2 equivalents after 20min exposure. 2. Analogous observations were made during serial additions of glycine, l-phenylalanine, l-leucine and l-lysine to preparations lacking the metabolic inhibitors and deficient in substrates needed for energy metabolism. In fresh cellular preparations the influx of glycine was then closely coupled to a stimulated flow of 2.1 equiv. of H(+) into the yeast. A similar number of K(+) ions left the cells. About 30% of the extra protons was subsequently ejected from the yeast. Deoxyglucose and antimycin together inhibited the ejection of protons. When the yeast had been fed with glucose energy metabolism was stimulated and almost as many protons as were absorbed with the amino acid were apparently ejected again. 3. Yeast preparations containing Na(+), instead of K(+), as the principal cation absorbed about 1 extra equivalent of H(+) after the addition of phenylalanine, glycine or leucine. This response was not observed in the presence of both deoxyglucose and antimycin. 4. The observations show that H(+) and, in certain circumstances, K(+) are co-substrates in the transport of the amino acids into the yeast. An analogy is drawn with the roles of Na(+) and K(+) as co-substrates in certain mammalian systems. The results lead to various models relating the physical flow of the co-substrate ions on the amino acid carrier to the transduction of chemical energy in an associated ion pump forming part of the mechanism for transporting amino acids into the yeast.

Biosynthesis of sphingolipid bases: IV. The biosynthetic origin of sphingosine in Hansenula ciferri

The synthesis and properties of 3-ketodihydrosphingosine-3- 14C(VIII) are described. Cell-free particulate fractions from the yeast Hansenula ciferri convert VIII exclusively to dihydrosphingosine; no 3-ketosphingosine or sphingosine could be detected. Similar fractions convert, 14C-ammonium palmitate to trams-2-hexadecenoic acid indicating that, in yeast, the biosynthetic pathways of saturated and unsaturated sphingolipids diverge at the fatty acyl CoA level. Substrate preferences for the enzyme(s) which mediates the condensation of serine with fatty acyl CoA to produce ketosphingolipid bases are: palmityl CoA > stearyl CoA > myristyl CoA > decanoyl CoA > lauryl CoA. Under appropriate conditions trans-2-hexadecenoyl CoA is an excellent substrate and, like palmityl CoA, leads to a mixture of sphingosine and dihydrosphingosine. α-Hydroxypalmityl CoA is not a substrate for the condensing enzyme and appears, therefore, not to be a precursor of phytosphingosine in yeast preparations. Most of the fatty acyl CoA derivatives tested are potent inhibitors of the condensing enzyme; bovine serum albumin is effective in reducing this inhibition.

Microbiological studies of Indonesian fermented foodstuffs.

Microbiological studies were made of certain Indonesian foodstuffs obtained from Malang, Surakarta, and Djakarta.Saccharomyces cerevisiae andCandida solani were isolated from ragi-roti (a baker's yeast preparation). From ragi-tempe and tempe were isolatedRhizopus oryzae, R. arrhizus, R. oligosporus, R. stolonifer, Mucor Rouxii, M. javanicus andTrichosporon pullulans. The microbiological flora of ragi-tape was found to includeCandida parapsilosis, C. melinii, C. lactosa sp. nov.,Hansenula subpelliculosa, H. anomala, H. malanga sp. nov.,Chlamydomucor oryzae andAspergillus oryzae. From ragi-ketjap, used to prepare soysauce, were isolatedRhizopus oligosporus, R. arrhizus, Aspergillus oryzae, andA. flavus, the latter species probably being an accidental contaminant. Two new species are described:Candida lactosa andHansenula malanga, from ragi-tape from Surakarta and Malang, respectively.

Interactions of uncoupling agent and of antimycin A with cytochrome b in yeast and heart muscle preparation under anaerobic conditions

When added to an anaerobic suspension of aerobically grown yeast, of mitochondria isolated from it, or of Keilin-Hartree heart muscle preparation, uncoupling agents elicited a considerable oxidation of cytochrome b. For a maximum effect, about three orders of magnitude higher concentrations were required than those effective in uncoupling oxidative phosphorylation, but an extrapolation to zero of the curve relating oxidation of cytochrome b to uncoupler concentration indicates that a slight effect may also occur at uncoupling concentrations. Ubiquinone was also partly oxidized, while cytochromes a·a 3 and c + c 1 remained unaffected. An unknown component of mitochondria may accept electrons from cytochrome b under these conditions. The effect was rather specific for uncoupling agents and was not displayed by a number of compounds devoid of uncoupling capacity. Antimycin A added to intact yeast cells or to isolated yeast mitochondria under anaerobic conditions also induced the anaerobic oxidation of cytochrome b. Under aerobic conditions, however, antimycin A prevented the cytochrome b oxidation via the respiratory chain and caused an additional slight reduction of cytochrome b to a form whose absorption maximum in the α-region was shifted to 565 nm. In the heart muscle preparation, antimycin A did not induce cytochrome b oxidation under anaerobic conditions but did cause an additional reduction by succinate of cytochrome b to the form absorbing at 565 nm. In normal ubiquinone-containing heart muscle particles, uncoupling agents did not induce the anaerobic oxidation of that part of cytochrome b which wits reducible by succinate in the presence of antimycin A. However, in ubiquinone-depleted particles, this part of cytochrome b became accessible to the uncoupler-induced anaerobic oxidation. The antimycin A block preventing oxidation of cytochrome b by air via the respiratory chain was relieved by some uncouplers at very high concentrations.

Anaerobic formation of ergosterol from a 5α-hydroxysterol by cell-free preparations of yeast

Anaerobic formation of ergosterol from a 5α-hydroxysterol by cell-free preparations of yeast

Rapid method for measuring extracellular water in yeast preparations.

A rapid procedure for the quantitative determination of extracellular water in bulk bakers' yeast was developed on the basis of the solute dilution principle. A reagent is prepared by synthesizing the diazonium ion of p-aminobenzoic acid and coupling it to peptone. This "azopeptone reagent" permits direct colorimetric measurement, which accounts for the rapidity and simplicity of the test. Potential errors due to osmotic effects are avoided by supplementing the reagent with saline and, more importantly, minimizing the duration of contact between reagent and cells. The new method has acceptable accuracy and precision, and may also be suitable for use with other microorganisms.

Enzymic polymerization of monosaccharides: II. Synthesis of 2-acetamido-2-deoxy-6-O-(α- d-galactopyranosyl)- d-glucose ( N-acetylmelibiosamine)

2-Acetamido-2-deoxy-6- O-(α- d-galactopyranosyl)- d-glucose ( N-acetylmelibiosamine) was synthesized by the catalytic action of an acetone-dried preparation of Saccharomyces carlsbergensis yeast on ( a) a mixture of d-galactose and N-acetyl- d-glucosamine, and ( b) a mixture of phenyl α- d-galactopyranoside and N-acetyl- d-glucosamine. A second disaccharide containing an amino sugar was isolated in reaction ( a), and its structure was tentatively established as 2-acetamido-2-deoxy-3- O-(α- d-galactopyranosyl)- d-glucose. The structure of N-acetylmelibiosamine was determined by selective periodate oxidation of the derived alcohol, and the similar oxidations of related model compounds are recorded.

Lysis of the cell walls of yeast ( Saccharomyces cerevisiae) by soil fungi

Two fungi, Fusarium sp. and an unidentified sterile fungus (no. 6), have been isolated from soil and their lytic activity towards cell walls of yeast (Saccharo-myces cerevisiae) examined. Growth of the two fungi on solid and liquid media containing autoclaved whole yeast or yeast cell walls resulted in lysis of the cell walls in both instances. Filtrates from cultures of both fungi grown on autoclaved walls, or whole yeast, showed glucanase activity when this was estimated with laminarin as substrate and this activity was greatest at pH 5·0. The cell walls of preheated whole yeast lost much of their opacity (phase-contrast microscopy) when incubated, in the presence of 2-mercaptoethanol at pH 5·0 and 7·5, with the culture filtrate from the sterile fungus no. 6. Isolated walls (unheated) were also incubated in a similar manner and chemical analysis showed that the walls had lost 77% (at pH 5·0) and 65% (at pH 7·5) of their anthrone-positive constituents after 6 days at 30 °C; the walls lost much of their opacity when incubated at pH 7·5 only. In the absence of 2-mercaptoethanol the losses amounted to 37% at pH 5·0 and 25% at pH 7·5. Ultra-thin sections of whole yeast and shadowed preparations of isolated cell walls examined in the electron microscope provided important information on the manner in which these materials were degraded.

Rapid Method for Measuring Extracellular Water in Yeast Preparations

A rapid procedure for the quantitative determination of extracellular water in bulk bakers' yeast was developed on the basis of the solute dilution principle. A reagent is prepared by synthesizing the diazonium ion of p-aminobenzoic acid and coupling it to peptone. This "azopeptone reagent" permits direct colorimetric measurement, which accounts for the rapidity and simplicity of the test. Potential errors due to osmotic effects are avoided by supplementing the reagent with saline and, more importantly, minimizing the duration of contact between reagent and cells. The new method has acceptable accuracy and precision, and may also be suitable for use with other microorganisms.

The Biosynthesis of β-Hydroxy-β-methylglutaryl Coenzyme A in Yeast: III. PURIFICATION AND PROPERTIES OF THE CONDENSING ENZYME THIOLASE SYSTEM

Abstract Attempts to separate β-hydroxy-β-methylglutaric acid coenzyme A-condensing enzyme and thiolase from yeast preparations by density gradient centrifugation, chromatography, and electrophoresis are described. In each case, it was not possible to prepare by these methods condensing enzyme free of thiolase activity. The results suggest that both proteins have closely similar physical properties. Each enzyme can be inactivated independently of the other by treatment with various reagents. Trypsin and chymotrypsin partially inactivate condensing enzyme. Papain, on the other hand, had no effect. The prior presence of acetoacetyl coenzyme A protects the condensing enzyme from inactivation by trypsin and chymotrypsin. Thiolase is completely inactivated by trypsin and chymotrypsin. The presence of CoA prior to treatment with trypsin partially protects thiolase from inactivation. Papain inactivates thiolase about 50% in the absence of substrates, but in the presence of acetoacetyl-CoA the inactivation goes rapidly to completion. Heat treatment inhibits condensing enzyme but not thiolase. The presence of acetyl-CoA protects condensing enzyme from heat inactivation and at the same time makes thiolase somewhat sensitive to heat. In agreement with previous results, iodoacetamide completely inhibits thiolase and only partially affects condensing enzyme. The presence of acetoacetyl-CoA or acetyl-CoA prior to the addition of iodoacetamide gives almost complete protection to condensing enzyme and partial protection to thiolase. CoA did not protect thiolase from inhibition. These results concerning the effect of substrates suggest that conformational changes in both condensing enzyme and thiolase are induced by the substrates, acetyl-CoA, acetoacetyl-CoA, and CoA. Kinetic measurements on condensing enzyme show that acetoacetyl-CoA exerts a large inhibitory effect. At low concentrations of acetyl-CoA, acetoacetyl-CoA appears to act as a competitive inhibitor. In the case of thiolase both acetoacetyl-CoA and CoA show potent substrate inhibition. Specificity studies with various acyl thioesters indicate that the thiolase activity studied differs from the β-ketothiolase and mixed thiolase studied by Stern and Drummond (4, 18).

On the question of biologically active fluorescent derivatives of thiamine.

The potassium permanganate and manganese dioxide oxidations of thiamine afford thiamine disulfide (83%) and thiochrone (17%). This mixture shows activity with respect to the decarboxylation of pyruvic acid in yeast preparations, and these components are apparently the fluorescent oxidation products of thiamine (quinochromes) that were discussed by Kinnersley. The decreased biological activity of the oxidation products arising from the reaction in 85% ethanol, as compared to 15% ethanol, is explained in terms of the dielectric constant of the solvent; solvents of high dielectric constants favor disulfide production, whereas solvents of low dielectric constants favor thiochrome formation.

Factors affecting fatty acid synthesis in cell-free preparations from [formula omitted

In earlier work from this laboratory, it was shown that fatty acid synthesis in yeast preparations is strongly influenced by carbon dioxide (Klein, 1957) and by a particulate fraction sedimentable from postmitochondrial supernatants (Klein and Booher, 1956; Klein, 1960; Abraham et al. , 1961; Den and Klein, 1961; Klein, 1963). Working with animal extracts, other workers have emphasized that citrate and other biochemical intermediates can exert profound effects on fatty acid formation, and several hypotheses based upon such interactions have been formulated (Vagelos et al. , 1963; Tubbs and Garland, 1963; Bortz and Lynen, 1963; Vagelos, 1964; Howard and Lowenstein, 1964). It is the purpose of this report to indicate that yeast preparations are also subject to controlling influences by certain intermediates of the oxidative and fermentative metabolism of glucose.

CONDENSATION OF ALPHA-KETOBUTYRATE AND ACETYL-COA IN BAKER'S YEAST.

Dialyzed cell-free preparations of baker's yeast fortified with magnesium and potassium ions, CoA and ATP incorporate 14C-labeled acetate in the presence of unlabeled α-ketobutyrate. This acetate-fixing reaction results in the formation of only one product that has been isolated by paper chromatography and is catalyzed by an enzyme which condenses in the absence of magnesium ions 1 mol of acetyl-CoA with 1 mol of α-ketobutyrate. The new condensing enzyme is very active in the crude extracts and has been separated by ammonium sulfate fractionation from other enzymes previously reported to occur in baker's yeast, which condense acetyl-CoA with the following α-ketoacids: glyoxylate, pyruvate, oxaloacetate, α-ketoisovalerate, and α-ketoglutarate.