PubMed Health⌕ Search

Biomedical subjects

E Cabib

Publications and source records attributed to E Cabib.

At least 55 records · Page 3Linked to original sources
PubMed1984

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.

Cell Wall↗
PubMed1983

Vectorial synthesis of a polysaccharide by isolated plasma membranes.

To ascertain the directionality of chitin synthesis by yeast plasma membranes, the external surface of Saccharomyces cerevisiae protoplasts was labeled with ferritin--concanavalin A. After protoplast lysis, plasma membranes were isolated and treated with trypsin to activate chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyl-transferase, EC 2.4.1.16). The membranes were then enrobed in agar and allowed to synthesize chitin from UDP-N-acetylglucosamine. After fixation and embedding in Epon, thin sections were stained for chitin with wheat germ agglutinin--colloidal gold complexes. The chitin marker was found near the ferritin-labeled external face of the membrane--i.e., the polysaccharide was located on the outside of the membrane, as it is in the intact cell. Chitin synthase activity was not detected in intact protoplasts before or after treatment with trypsin. The enzyme became available to trypsin activation after lysis of the protoplasts. Together with similar, previously reported experiments on the inactivation of chitin synthase by glutaraldehyde, these results indicate that the enzyme faces the interior of the cell. We conclude that, both in vivo and in vitro, the synthase receives N-acetylglucosamine residues from UDP-N-acetylglucosamine at the cytoplasmic face of the membrane and transfers them vectorially to a growing chain of chitin that is concomitantly extruded to the outside.

Cell Membrane↗
PubMed1983

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

Cell Cycle↗
PubMed1982

Interaction between yeast beta-(1 goes to 3)glucan synthetase and activating phosphorylated compounds. A kinetic study.

Yeast beta-(1 goes to 3)glucan synthetase is stimulated by ATP or GTP. The structural requirements for the activation were investigated by testing several phosphorylated compounds. The simplest substance with stimulatory ability was inorganic pyrophosphate. Addition of a nucleoside, as in GDP, decreased the concentration required for half-maximal stimulation; a third phosphate group, as in GTP, further enhanced the stimulatory capacity. On the other hand, esterification of the terminal phosphate of GTP with a nucleoside or a methyl group led to a total loss of activating ability: dinucleoside triphosphates and the gamma-phosphate methyl ester of GTP acted as competitive antagonists of the activators. alpha,beta- and beta,gamma-imino and -methylene derivatives of both ATP and GTP stimulated the enzymatic activity, suggesting that activation can occur without covalent transfer either of the terminal phosphate or pyrophosphate, or of the nucleotidyl residue. The stimulatory effect of the beta,gamma-imino derivatives of ATP and GTP was not additive. The inhibition constants obtained with gamma-phosphate esters of GTP were the same for either one of the two imino analogs. It is concluded that adenosine and guanosine derivatives bind to the same domain of the enzyme. It is also postulated that activators may interact with the enzyme or with a regulatory protein at two locations, a binding site for the nucleoside moiety and a "functional" site for the pyrophosphate residue.

Enzyme Activation↗
PubMed1982

Endochitinase, a mannan-associated enzyme from Saccharomyces cerevisiae.

A chitinase was extracted with digitonin from intact yeast cells and purified by adsorption-digestion on chitin. The purified enzyme liberates oligosaccharides of various sizes from chitin, thus behaving as an endochitinase. As found with other chitinases, the yeast enzyme is much more active on nascent chitin, i.e. the chitin formed in the same reaction mixture by the corresponding synthetase, than on preformed polysaccharide. The enzyme has a very low pH optimum, about pH 2.5, and is quite stable at pH 3. Polyacrylamide gel electrophoresis of different preparations of purified chitinase revealed a variable number of protein bands, whose pattern often changed after storage of the enzyme. The distribution of activity in the gel matched that of the stainable material. Association of the chitinase with mannan is indicated by the following results: (a) coincidence in Coomassie blue-staining and periodate-Schiff-staining bands after disc gel electrophoresis; (b) adsorption of the activity on concanavalin A-Sepharose and elution with alpha-methylmanoside; (c) precipitation of the chitinase with appropriate antimannan sera. A carbohydrate content of approximately 18% was found in a trichloroacetic acid-precipitated sample of purified enzyme. Protein and mannan were not dissociated by boiling with sodium dodecyl sulfate, urea, and beta-mercaptoethanol. It could not, however, be conclusively established whether protein and carbohydrate are covalently linked, because the chitinase is resistant to endo-beta-N-acetyl-glucosaminidase.

Chitinases↗
PubMed1982

Secretory character of yeast chitinase.

During the conversion of yeast cells to protoplasts, about half of the yeast chitinase was liberated into the medium, an indication that this portion of the enzyme is located in the periplasmic space. At least part of the remaining chitinase appears to be enclosed in vacuoles or in vesicle that co-purify with them, as indicated by a 14-fold enrichment of the enzymatic activity in a vacuole fraction isolated from a protoplast lysate. When protoplasts were incubated in growth medium, part of the chitinase was liberated in growth medium, part of the chitinase was liberated into the medium. It is concluded that yeast chitinase is a secretory enzyme, like invertase and acid phosphatase. The enzyme appears to be stored in vesicles as a prelude to its secretion into the periplasmic space. The possible function of yeast chitinase in cell division is discussed.

Cell Division↗
PubMed1982

Activation of chitin synthetase in permeabilized cells of a Saccharomyces cerevisiae mutant lacking proteinase B.

Digitonin treatment at 30 degrees C of a Saccharomyces cerevisiae mutant lacking proteinase B permeabilized the cells and caused rapid and extensive activation of chitin synthetase in situ. The same result was obtained with a mutant generally defective in vacuolar proteases. By lowering the temperature and using different permeabilization procedures, we showed that increases in permeability and activation are distinct processes. Activation was inhibited by the protease inhibitors antipain and leupeptin, but by pepstatin or chymostatin. Metal chelators were also inhibitory, and their effect was reversed by the addition of Ca2+ but not by Mg2+. Antipain added together with Ca2+ after incubation of the cells in the presence of a chelating agent prevented reversal of inhibition, a result that was interpreted as indicating that antipain acts either on the same step affected by Ca2+ or on a subsequent step. Efforts to obtain activation in cell-free extracts were unsuccessful, but it was possible to extract the synthetase, once activated, by breaking permeabilized cells with glass beads. Treatment of the cell-free extracts with trypsin led not only to increased activity of chitin synthetase, but also to a change in the pH-activity curve and a diminished requirement by the enzyme for free N-acetylglucosamine. These observations suggest that the modification undergone by the synthetase during endogenous activation is different from that brought about by trypsin treatment.

Cell Membrane Permeability↗
PubMed1980

Biosynthesis of the yeast cell wall. I. Preparation and properties of beta-(1 leads to 3)glucan synthetase.

The synthesis of the major linkage found in yeast cell wall structural polysaccharides, glucosyl-beta-(1 leads to 3)-glucosyl, was studied with a membrane preparation from Saccharomyces cerevisiae. The sugar donor was UDP-glucose, and the reaction required addition of glycerol bovine serum albumin, and ATP or GTP for maximal activity. Under optimal conditions, extremely efficient glucose transfer was obtained, with 20 to 50% of the substrate utilized in 20 min at 30 degrees C. The polysaccharide formed in the reaction was insoluble in water and soluble in alkali; it was characterized enzymatically and chemically as a beta-(1 leads to 3)-linked linear glucan of chain length 60 to 80. The terminal reducing group was found to be labeled with 14C, as was the substrate used; therefore, the polysaccharide is synthesized de novo. For each glucosyl group transferred, one equivalent of UDP was formed. No evidence was found for a lipid-linked intermediate. When yeast protoplast lysates were subjected to fractionation by centrifugation in Renografin gradients, glucan synthetase was found in the plasma membrane fraction, with the same distribution and sidedness as chitin synthetase. Because of the spatially restricted growth of the cell wall during cell division in budding yeasts, this result suggests localized and reversible activation of the enzyme during the cell cycle.

Cell Membrane↗
PubMed1980

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study.

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.

Cell Wall↗
PubMed1979

Chitin synthetase distribution on the yeast plasma membrane.

Purified, intact yeast plasma membranes were allowed to synthesize chitin, and the nascent chains of polysaccharide were observed either by the fluorescence produced with a brightener or by autoradiography. By both methods, it was concluded that the newly formed chitin emerged at many sites on each membrane. Thus, the synthetase that catalyzes chitin formation has a similar distribution. Since chitin synthetase is found mainly in a zymogen form, these results confirm the hypothesis that initiation of the chitinous primary septum of Saccharomyces occurs by localized activation of the uniformly distributed zymogen.

Cell Membrane↗
PubMed1978

Solubilization and partial purification of yeast chitin synthetase. Confirmation of the zymogenic nature of the enzyme.

Chitin synthetase was solubilized with digitonin from a particulate yeast fraction. The solubilized enzyme, which did not sediment at 200,000 X g and emerged after the void volume in a Sepharose 6B column, was active only after treatment with a protease. This confirms that chitin synthetase exists in the plasma membrane as a zymogen and that initiation of the chitin septum occurs by localized activation of the enzyme. By differential extraction with sodium cholate and digitonin, followed by chromatography on Sepharose 6B, a 20-fold purification of the enzyme was achieved with respect to the crude particles. The purified enzyme showed a requirement for a phospholipid; phosphatidylserine and lysophosphatidylserine were the best activators. Unsaturated fatty acids strongly inhibited synthetase activity, whereas their saturated counterparts were inert. The solubilized enzyme catalyzed the formation of insoluble chitin in the absence of added primer. The synthetic polysaccharide was examined by electron microscopy and found to consist of lozenge-shaped particles about 60 nm long and 10 nm wide.

Chitin Synthase↗