[Inhibiting effect of concanavalin A on certain biosynthetic processes in spheroplasts of the yeast Saccharomyces cerevisiae].
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The effect of concanavalin A on biosynthesis of nucleic acids, proteins, structural polysaccharides and glycoproteins of the yeast cell membrane and of enzymes having different localization in the cell as well as on other processes occurring in spheroplasts of the yeasts Saccharomyces cerevisiae IBPhM-350 and CCY 21-4-13 were studied. In both yeast strains lectin strongly inhibited total protein synthesis and produced a weaker inhibiting effect on DNA and RNA synthesis. This was accompanied by a decrease of the activity of the majority of already known enzymes (acid phosphatase, invertase, alpha-glucosidase, polyphosphatase, pyrophosphatase, ATPase) and glucose consumption. In addition concanavalin A inhibited the synthesis of structural components of the yeast cell membrane, i.e. mannane and glucane. The data obtained suggest that lectin (50 microgram/ml or higher) has a toxic effect on yeast spheroplasts (or protoplasts).Keywords:
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Mannan
Protoplast
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Candida utilis, Saccharomyces cerevisiae, S. fragilis, Pichia polymorpha , and Hansenula anomala yeast cells, harvested in the early logarithmic phase, were attacked with purified β(1–3)-glucanase from Micromonospora chalcea , which resulted in the liberation of protoplasts. The treated cells were observed under the electron microscope before the protoplasts were liberated. Differences in the cell walls of the enzyme-treated and untreated cells were observed. The action of the glucanase was also tested against isolated walls of C. utilis . The enzyme attacked the S. cerevisiae cell wall in a uniform manner. The attack on S. fragilis was located in certain zones of the cell wall, where breakage occurred and through which the protoplast emerged. On the other three yeasts, an intermediate attack was observed, not as definitely located as in S. fragilis , yet less uniformly than in S. cerevisiae .
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Lytic cycle
Glucanase
Spheroplast
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A cell-wall-surface protein purified from the cells of Saccharomyces cerevisiae NCYC 227 was found to be involved in the non-sexual flocculation of this yeast. This 13 kDa protein was found to bind specifically to mannose. The protein bound to mannans isolated from yeast as well as in situ to intact cells, but only in the presence of calcium ions. The protein, a mannoprotein, formed aggregates as revealed in SDS-PAGE. Urea and higher temperatures prevented protein aggregation, suggesting that the flocculation of S. cerevisiae is primarily due to hydrogen bonding between mannan and protein.
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The effect of concanavalin A on biosynthesis of nucleic acids, proteins, structural polysaccharides and glycoproteins of the yeast cell membrane and of enzymes having different localization in the cell as well as on other processes occurring in spheroplasts of the yeasts Saccharomyces cerevisiae IBPhM-350 and CCY 21-4-13 were studied. In both yeast strains lectin strongly inhibited total protein synthesis and produced a weaker inhibiting effect on DNA and RNA synthesis. This was accompanied by a decrease of the activity of the majority of already known enzymes (acid phosphatase, invertase, alpha-glucosidase, polyphosphatase, pyrophosphatase, ATPase) and glucose consumption. In addition concanavalin A inhibited the synthesis of structural components of the yeast cell membrane, i.e. mannane and glucane. The data obtained suggest that lectin (50 microgram/ml or higher) has a toxic effect on yeast spheroplasts (or protoplasts).
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Mannan
Protoplast
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beta-Glucanases secreted into culture fluid by protoplasts or intact cells of the yeast Saccharomyces cerevisiae were investigated for the presence of covalently linked carbohydrates. Gel filtration of the enzymes on Biogel A-1.5m showed that endo-beta-1,3-glucanase is a polydisperse enzyme of high-molecular weight which elutes in about the same volume as external yeast invertase. Exo-beta-glucanase was eluted from the gel as a much lighter enzyme. Endo-beta-1,3-glucanase added to a mixture of extracellular mannoproteins was precipitated by concanavalin A to a similar extent to mannan, invertase and acid phosphatase. Under the same conditions exo-beta-glucanase did not interact with the lectin, but was partially precipitated from the solution in the absence of foreign mannan or mannan-proteins. The results show that endo-beta-1,3-glucanase of S. cerevisiae is a mannoprotein of a similar nature to external invertase and acid phosphatase. However, exo-beta-glucanase appears to be a glycoprotein which does not contain the highly branched mannan polymer in its molecule.
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Schlenk , F. (Argonne National Laboratory, Argonne, Ill.), and J. L. Dainko . Action of ribonuclease preparations on viable yeast cells and spheroplasts. J. Bacteriol. 89: 428–436. 1965.—Recrystallized pancreatic ribonuclease releases more than half of the ultraviolet-absorbing cellular constituents of Candida utilis and Saccharomyces cerevisiae . The presence of salts and buffer interferes with the action of the enzyme on the cytoplasmic membrane, and intracellular constituents show a similar inhibitory effect. Polymerized ribonuclease and conformational isomers cannot penetrate the pores of the yeast cell wall, but they are effective on the membrane of spheroplasts. All forms of the enzyme interfere strongly with the viability of the cells.
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Pancreatic ribonuclease
Cell membrane
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Abstract Yeast flocculation involves the binding of surface lectins on flocculent yeasts, to carbohydrate receptors present as constituents of yeast cell walls. Receptors were investigated by coflocculation of flocculent strains of Saccharomyces cerevisiae , both Flo 1 and NewFlo phenotypes, to known mnn mutants which vary in the wall mannan structure. Strong coflocculation was found with mnn1 , mnn4 , mnn9 and control strains, while very little coflocculation was found with mnn2 and mnn5 strains. In constrast, aggregation of these muatants by concanavalin A, a lectin with similar sugar inhibition to NewFlo phenotype flocculation, showed strong aggregation of mnn1 , mnn4 , and mnn5 strains and poor aggregation of mnn2 and mnn9 strains. The mmn mutant data suggested that flocculation receptorss were the outer‐chain mannan side‐branches, two or three mannose residues in length, confirming an earlier theory based on sugar inhibition data. The similarities and differences between flocculation and concanavalin A aggregation are discussed.
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Yeast Saccharomyces cerevisiae is the most significant source of enzyme invertase. It is mainly used in the food industry as a soluble or immobilized enzyme. The greatest amount of invertase is located in the periplasmic space in yeast. In this work, it was isolated into two forms of enzyme from yeast S. cerevisiae cell, soluble and cell wall invertase (CWI). Both forms of enzyme showed same temperature optimum (60°C), similar pH optimum, and kinetic parameters. The significant difference between these biocatalysts was observed in their thermal stability, stability in urea and methanol solution. At 60°C, CWI had 1.7 times longer half-life than soluble enzyme, while at 70°C CWI showed 8.7 times longer half-life than soluble enzyme. After 2-hr of incubation in 8 M urea solution, soluble invertase and CWI retained 10 and 60% of its initial activity, respectively. During 22 hr of incubation of both enzymes in 30 and 40% methanol, soluble invertase was completely inactivated, while CWI changed its activity within the experimental error. Therefore, soluble invertase and CWI have not shown any substantial difference, but CWI showed better thermal stability and stability in some of the typical protein-denaturing agents.
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Hansenula mrakii secretes extracellularly a killer toxin which kills sensitive Saccharomyces cerevisiae. In protoplasts of this yeast, the killer toxin selectively inhibited the synthesis of alkali-insoluble acid-insoluble polysaccharides consisting mainly of beta-glucan, but did not inhibit either the synthesis of other cell wall polysaccharides, such as mannan, chitin and alkali-insoluble acid-soluble polysaccharides, or the synthesis of protein. Consistent with these results, the toxin was inhibitory to the beta-(1,3)-glucan synthetase activity of a cell-free extract from sensitive S. cerevisiae.
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