Showing papers on "Yeast published in 1973"

The structure of a β-(1→3)-d-glucan from yeast cell walls

Abstract: By selective enzymolysis, or chemical fractionation, a minor polysaccharide component has been isolated from yeast (Saccharomyces cerevisiae) glucan. This minor component has a degree of polymerization of about 130-140, a highly branched structure, and a high proportion of beta-(1-->6)-glucosidic linkages. The molecules also contain a smaller proportion of beta-(1-->3)-glucosidic linkages that serve mainly as interchain linkages, but some may also be inter-residue linkages.

The structure of a beta-(1 leads to 3)-D-glucan from yeast cell walls.

Abstract: Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched beta-(1-->3)-glucan of high molecular weight (about 240000) containing 3% of beta-(1-->6)-glucosidic interchain linkages. The minor component is a branched beta-(1-->6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major beta-(1-->3)-glucan component may vary considerably in degree of branching.

Cytochrome c: a thermodynamic study of the relationships among oxidation state, ion-binding and structural parameters. 1. The effects of temperature, pH and electrostatic media on the standard redox potential of cytochrome c.

Abstract: The standard redox potential at I= 0.01, 25°C and pH 7.0 has been determined for the following species of cytochrome c: horse heart, baker's yeast isoenzyme-1, Candida species yeast, tuna heart and turkey heart. The thermodynamic parameters of the redox reaction were evaluated and found to be identical, within experimental error, in the five species investigated. The effect of pH on the standard redox potential of horse heart cytochrome c was studied in the pH range 7.0–11.2. A single heme-linked ionization of the oxidized protein was observed in this pH range, with a dissociation constant of 3 × 10−9 at I= 0 and 25°C. The effects of the electrostatic media on the standard redox potential of horse heart cytochrome c depend on the nature of the anions employed. For non-binding medium at 25°C, the observed potential dependence on the ionic strength is given by the equation: E0obs= 0.274–0.336 √I/(1 + 6√I). In binding medium, specific binding of ions to the protein takes place.

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

Abstract: 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 (Km=1.6mm). The uptake of protons was also faster in the presence of α-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, α-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.

Assay of yeast enzymes in situ. A potential tool in regulation studies.

Abstract: A permeabilization method which allows the assay of several intracellular enzymes within the boundaries of the yeast cell wall is described. The kinetic properties of hexokinase and pyruvate kinase examined in the permeabilized cells, including the allosteric activation of the latter by fructose bisphosphate, are essentially the same as in cell-free extracts.

Isolation and Characterization of a Thermotolerant Methanol-Utilizing Yeast

Abstract: A yeast capable of growth on methanol as its sole carbon-energy source was isoalted from soil samples and identified as a strain of Hansenula polymorpha. A continuous enrichment culture at 37 C with a simple mineral salts medium was used to select this organism. The isolate, designated DL-1, has a maximal specific growth rate of 0.22 per h, at pH 4.5 to 5.5 and temperatures of 37 to 42 C, in simple mineral salts medium with methanol (0.5%), biotin, and thiamine. Growth occurred in a chemostat at temperatures up to 50 C, with strong growth at 45 C. The maximal growth yield of the yeast on methanol was 0.36 g of dry cell weight per g of methanol, and the yield on oxygen was 0.37 g of dry cell weight per g of O2. Protein content of the isolate is 46%, and total nucleic acid content varies from 5.0 to 7.0% with increasing growth rate from 0.08 to 0.20 per h. The amino acid profile of this yeast protein indicates that it could serve as a good source of food protein. Feeding studies with rats show the yeast to have no toxic effects.

Properties of a Yeast Cytochrome P‐450‐Containing Enzyme System which Catalyzes the Hydroxylation of Fatty Acids, Alkanes, and Drugs

Abstract: The growth of Candida tropicalis on tetradecane causes the induction of a cytochrome P-450-containing enzyme system which catalyzes the hydroxylation of fatty acids, hydrocarbons and drugs. When the cells are broken by treatment with a French pressure cell, the cytochrome P-450 is obtained in an apparently soluble form. The enzyme system was resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and a heat-stable lipid fraction, all of which are necessary, along with NADPH and molecular oxygen, for the conversion of laurate to ω-hydroxylaurate. NADH alone is almost completely inactive but causes a doubling of the activity when present along with a saturating level of NADPH. The yeast reductase and lipid fractions may be replaced by corresponding fractions obtained from rat liver microsomes. The effect of various phospholipids on the hydroxylation activity was investigated, and a yeast lysophosphati-dylethanolamine fraction was shown to be most effective as judged by laurate hydroxylation. The yeast cytochrome P-450 is not readily autoxidizable, as shown by experiments in which it was reduced extensively by NADPH in the presence of phospholipid and the reductase under aerobic conditions.

Identification of the Lipid Intermediate in Yeast Mannan Biosynthesis

Abstract: The unsaponifiable lipid fraction of Saccharomyces cerevisiae contains a phosphorylated component which accepts a mannosyl residue from GDP-mannose and transfers it to growing mannan chains. Both reactions are catalyzed by membrane-bound enzymes. The lipid component has been purified more than 1000-fold (based on lipid-bound phosphate). After dephosphorylation with an orthophosphoric monoester phosphohydrolase this acceptor was shown by mass spectroscopy to be identical with authentic yeast dolichol. The “lipid” intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol. The “lipid” intermediate intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol monophosphate. Yeast dolichol is a mixture of five homologous polyprenols with saturated α-isoprene units, as shown by Dunphy et al. in 1967. All these homologues seem also to be present in the cells as monophosphates. As compared to free dolichols the phosphorylated forms only amount to about 10–20%.

Pantetheine‐Free Mutants of the Yeast Fatty‐Acid‐Synthetase Complex

Abstract: Among 52 Saccharomyces cerevisiae fatty acid synthetase (fas)mutants screened for their ability to incorporate 14C-labeled pantothenic acid into the fatty acid synthetase multienzyme complex, especially those of fas-complementation group VII had lost this ability. The purified fatty acid synthetase complexes of all the 19 fas-mutants available from this group were shown to be free of 14C-labeled pantothenic acid. The amount of pantothenate incorporated into the enzymes of several other fas-mutants of the non-pleiotropic complementation groups II, V, VI and VIII, however, was similar to that observed with wild-type fatty acid synthetase. The purified pantothenate-free fatty acid synthetases of five different group VII fas-mutants have been tested for the seven known component enzymes of the complex. In all mutants, only the β-ketoacyl synthetase was inactive whereas in vitro all the other partial activities were unimpaired. By sodium dodecylsulfate-polyacrylamide-gel electrophoresis, no differences could be observed between the protein structure of the pantothenate-free mutant fatty acid synthetase and that of the wild-type comp ex. Both were separated into three different components A, B and C with molecular weights of 185000, 180000 and 177000, respectively. However, some fas-mutants consist only of the components A and B, others only of B and C. The study of various [14C]-pantothenate-labeled mutant fatty acid synthetases suggests that component C originates from A presumably by limited proteolysis. In the undegraded complex AB, [14C]pantothenate is only associated with the component A. Since in one of the mutants studied A is completely converted to C, it is concluded that A is one distinct component rather than a group of components with identical molecular weights. It is tentatively suggested that the gene product of fas 2, one of the two known fatty-acid-synthetase gene clusters in yeast, is only one, single and multifunctional polypeptide chain. Therefore, it appears that in yeast, the acyl carrier protein is not an individual protein component of the fatty acid synthetase complex, but only a distinct region of the multifunctional polypeptide chain encoded by fas 2.

Yeast growth in relation to the dissolved oxygen and sterol content of wort

Abstract: The extent of the requirement for oxygen in cells of brewing yeast is determined by the availability of oxygen during propagation. Cells with no oxygen requirement ferment satisfactorily when added to either air-saturated or de-aerated wort. Cells produced during fermentation develop an oxygen-requirement and ferment poorly when added to de-aerated wort because of restriction of both rate and extent of exponential growth. The quantity of dissolved oxygen needed to ensure satisfactory growth varies greatly with yeast strain. In all cases examined, the oxygen requirement can be eliminated by addition to the growth medium of ergosterol and Tween 80 However Tween 80 alone is without effect. It seems likely that oxygen is required because it is essential for biosynthesis of sterols.

Strains of yeast lethal to brewery yeasts

Abstract: Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.

10 3-Phosphoglycerate Kinase

Abstract: Publisher Summary This chapter describes 3-phosphoglycerate kinase. The enzyme 3-phosphoglycerate kinase was isolated from yeast. The crystalline yeast preparation has been the principal supply of the enzyme. 3-Phosphoglycerate kinase catalyzes the transfer of “energy-rich” phosphate from the acid anhydride bond of 1,3-DPGA to the terminal phosphate of adenosine diphosphate (ADP). Divalent cations are essential to the reaction because the magnesium complexes of the nucleotides are most likely the true substrates. 3-Phosphoglycerate kinase is involved in carbon fixation in many plant tissues; it is the enzyme reacting immediately after one of the primary carbon fixing enzymes, ribulosediphosphate carboxylase. 3-Phosphoglycerate kinase has been shown to occur in a variety of higher plants and in blood, but it exists in highest concentrations in yeast, about 2 mg/g wet weight. Because of compartmentalization within the cells, it probably exists at concentrations up to four times greater than these that is up to 0.1 mM. In this chapter, biological behavior of phosphoglycerate kinase is described. Isolation and molecular properties are explained. Reaction kinetics is also discussed in the chapter.

Very Long-Chain Fatty Acids in Yeast

Abstract: Novel fatty acids ranging from 20 to 30 carbons have been found in Saccharomyces cerevisiae. These comprise 1 to 2% of the total fatty acid fraction.

Enzyme activities associated with carbohydrate synthesis and breakdown in the yeast and mycelial forms of Candida albicans.

Abstract: SUMMARY: The mycelial and blastospore forms of Candida albicans have been grown under conditions in which the only environmental variable was temperature. The activity of phosphoglucose isomerase, phosphofructokinase and the first enzyme of the hexose-monophosphate pathway and the pathways for chitin and mannan synthesis has been determined in extracts at different times in the cell cycle. Phosphofructokinase has been partially purified from both forms and the effect of adenosine phosphates on its activity determined. In this yeast, concentrations of glucose-6-phosphate, fructose-6-phosphate, ATP, ADP and AMP differed in the two growth forms and at different times in the growth cycle. L-Glutamine-D-fructose-6-phosphate aminotransferase activity at concentrations of fructose-6-phosphate found in the cell was appreciably greater in mycelium than in blastospores. The metabolism of [14C]glucose through the hexose monophosphate pathway was low after 4 and 18 h growth, and considerably increased at 10 h. The results support the concepts of a requirement for NADPH for cell division and suggest that control over the synthesis of chitin and mannan may, in part, be provided through control of the activity of phosphofructokinase by adenosine phosphates.

Morphological and Structural Changes During the Yeast-to-Mold Conversion of Phialophora dermatitidis

Abstract: The details of the morphological and structural events occurring during yeast-to-mold conversion of the human pathogenic fungus Phialophora dermatitidis as seen by phase-contrast microscopy and electron microscopy are described and illustrated. Budding yeasts growing exponentially were observed to have thin walls and a cytoplasm exhibiting the characteristics of rapidly growing cells including numerous mitochondria, abundant ribosomes, few vacuoles, and little accumulation of storage material. In contrast, thick-walled yeasts were characterized by less apparent or significantly fewer mitochondria and ribosomes and the presence of considerable amounts of storage materials. Microscope observations of yeast-to-mold conversion revealed that only thick-walled yeasts having prominent lipid bodies in their cytoplasm converted to hyphal forms. Typically, the thick-walled yeast formed two to a number of moniliform hyphal cells which in turn often produced true hyphae. The results indicated that yeasts of P. dermatitidis must acquire spore-like characteristics by becoming thick-walled and by accumulating considerable endogenous substrate reserves before they convert and produce hyphae.

Some methods for processing of single-cell protein.

Abstract: Methods for the production of protein concentrates, with a low content of nucleic acid, in kilogram quantities from yeast have been studied with the aid of equipment designed for operation on pilot-plant scale. The influence of drum drying and mechanical disintegration on the nutritive value of the yeast was also investigated. Drum drying and mechanical disintegration improved the nutritive value of the yeast but high extractability of protein and nucleic acid was only obtained after mechanical disintegration. Protein concentrates without and with cell walls were produced from mechanically disintegrated yeast. The different fractions which were obtained when separating cell walls and precipitating protein by heating at alkaline pH, were analyzed. After protein precipitation, about 90% of the RNA could be precipitated from the supernatant by addition of acid, giving a product containing 50% RNA of the dry weight. The protein precipitate obtained after cell wall separation had an RNA content of less than 2% and contained 70–l75% of the amino acids in the starting yeast material. Protein concentrates containing cell walls were produced by precipitating protein by heating at alkaline pH directly after mechanical disintegration. The content of RNA was about 2% and the yield of amino acids was 70–80%. It was found that the nutritive value of the protein concentrate was higher than that of the starting yeast material. To produce such a protein concentrate on a large scale, the process described can probably be employed.

Studies on yeast hemoglobin. The properties of yeast hemoglobin and its physiological function in the cell.

Abstract: Among the Candida species, C. mycoderma and C. roubsta possess significant amounts of a hemoglobin-like pigment. This pigment is identified as yeast hemoglobin by its spectroscopic properties and its ability of reversible binding with oxygen. The oxygen affinity of yeast hemoglobin in the respiring and nonrespiring cells is 0.02 μM which is identical to the value observed with an isolated yeast hemoglobin. Yeast hemoglobin in the cell can be decomposed specifically by pretreatment of the cells with ethyl hydrogen peroxide. Comparison of the hemoglobin-free cells with the intact cells indicates that a lack of yeast hemoglobin does not cause any significant delay of cell multiplication. Neither the rate of cell respiration nor the behaviors of the redox states of mirochondrial cytochromes responding to the changes of oxygen concentration between 1 and 0.01 μM is affected by the presence or absence of yeast hemoglobin.

The effect of substitution at C-2 of d-glucose 6-phosphate on the rate of dehydrogenation by glucose 6-phosphate dehydrogenase (from yeast and from rat liver)

Abstract: 1. The deoxyfluoro-d-glucopyranose 6-phosphates are substrates for both yeast and rat liver glucose 6-phosphate dehydrogenase. 2. The V max. values (relative to d-glucose 6-phosphate) were determined for a series of d-glucose 6-phosphate derivatives substituted at C-2. The V max. values decreased with increasing electronegativity of the C-2 substituent. This is consistent with a mechanism involving hydride-ion transfer. 3. 2-Deoxy-d- arabino -hexose 6-phosphate (2-deoxy-d-glucose 6-phosphate) showed substrate inhibition with the yeast enzyme but not with the rat liver enzyme. 4. 2-Amino-2-deoxy-d-glucose 6-phosphate (d-glucosamine 6-phosphate) was a substrate for the yeast enzyme but a competitive inhibitor for the rat liver enzyme. 5. Lineweaver–Burk plots for the d-glucose 6-phosphate derivatives with yeast glucose 6-phosphate dehydrogenase were biphasic.

Subcellular Localization of the Leucine Biosynthetic Enzymes in Yeast

Abstract: When baker's yeast spheroplasts were lysed by mild osmotic shock, practically all of the isopropylmalate isomerase and the beta-isopropylmalate dehydrogenase was released into the 30,000 x g supernatant fraction, as was the cytosol marker enzyme, glucose-6-phosphate dehydrogenase. alpha-Isopropylmalate synthase, however, was not detected in the initial supernatant, but could be progressively solubilized by homogenization, appearing more slowly than citrate synthase but faster than cytochrome oxidase. Of the total glutamate-alpha-ketoisocaproate transaminase activity, approximately 20% was in the initial soluble fraction, whereas solubilization of the remainder again required homogenization of the spheroplast lysate. Results from sucrose density gradient centrifugation of a cell-free particulate fraction and comparison with marker enzymes suggested that alpha-isopropylmalate synthase was located in the mitochondria. It thus appears that, in yeast, the first specific enzyme in the leucine biosynthetic pathway (alpha-isopropylmalate synthase) is particulate, whereas the next two enzymes in the pathway (isopropylmalate isomerase and beta-isopropylmalate dehydrogenase) are "soluble," with glutamate-alpha-ketoisocaproate transaminase activity being located in both the cytosol and particulate cell fractions.

New developments in mutagenicity screening techniques with yeast.

Abstract: Yeasts provide a useful and versatile tool for studying genetic phenomena. The genetics of Sacchlaromyces cerevisiae has been intensively studied for many years, and as a result considerable information has been accumulated. Excellent current and comprehensive reviews on the genetics of yeast are available (1, 2), including one that is particularly directed toward studies of deleterious genetic effects of chemicals (3). Yeasts are eukaryotic organisms and therefore contain a nucleus and cytoplasm containing various other differentiated organelles quite similar to other higher life forms (4). The genetic apparatus consists of at least 17 chromosomes in the haploid S. cerevisiae identified by genetic mapping (5). Thus the structural organization of the genetic information is analogous to that in other higher life forms. Equally important is the well-known life cycle in yeast which, genetically, provides for classical mitotic and meiotic functions such as those existing in differentiated cells of more complex multicellular organisms. Yeasts, as well as all higher life forms, contain extrachromosomal DNA which provides essential genetic in-