Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Yeast protoplasts were coated with different amounts of concanavalin A. Upon subsequent lysis and centrifugation in isopycnic density gradients, it was found that the buoyant density of plasma membranes was progressively increased from 1.125 to 1.21, according to the amount of bound concanavalin A. Enzymes that are attached to the plasma membrane showed the same density modifications and could thus be distinguished from constituents of intracellular membranes and organelles. With this methodology, it was confirmed that about two-thirds of yeast chitin synthetase is associated with the plasma membrane. The remainder of the enzyme was found in a peak at a lower density. Vanadate-sensitive ATPase showed a similar pattern, whereas GDP-mannose dolichyl-phosphate mannosyltransferase, an enzyme attached to the endoplasmic reticulum, remained in the same position in the gradients, irrespective of the amount of concanavalin A associated with the plasma membrane. Potential applications of this technique to the determination of plasma membrane markers and to the separation of subcellular organelles are discussed.
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PMID:Modification of yeast plasma membrane density by concanavalin A attachment. Application to study of chitin synthetase distribution. 241 28

Plasma membrane ghosts were isolated from Candida albicans ATCC 10261 yeast cells following stabilisation of spheroplasts with concanavalin A, osmotic lysis and Percoll density gradient centrifugation. Removal of extrinsic proteins with NaCl and methyl alpha-mannoside gave increased ATPase and chitin synthase specific activities in the resultant plasma membrane fraction. Sonication of this fraction yielded unilamellar plasma membrane vesicles which exhibited ATPase and chitin synthase specific activities of 4.5-fold and 3.0-fold, respectively, over those of the plasma membrane ghosts. ATPase activity in the membrane ghosts was optimal at pH 6.4, showed high substrate specificity (for Mg X ATP) and was inhibited 80% by sodium vanadate but less than 4% by oligomycin and azide. The effects of a range of other inhibitors were also characterised. Temperature effects of ATPase activity were marked, with a maximum at 35 degrees C. Breaks in the Arrhenius plot, at 12.2 degrees C and 28.9 degrees C, coincided with endothermic heat flow peaks detected by differential scanning calorimetry. ATPase was solubilised from the plasma membranes with Zwittergent in the presence of glycerol and phenylmethylsulphonyl fluoride and partially purified by glycerol density gradient centrifugation. The solubilised enzyme hydrolysed Mg X ATP at Vmax = 20 mumol X min-1 X mg-1 in the presence of phospholipids, with optimal activity at pH 6.0--6.5.
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PMID:The isolation of plasma membrane and characterisation of the plasma membrane ATPase from the yeast Candida albicans. 293 95

We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations. These procedures were used to examine the subcellular distribution of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast. Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters. One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter = 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-beta-glucan synthetase activity. The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase. This fraction cosedimented with the main peak of 1,3-beta-glucan synthetase. A third, minor population of chitin synthetase particles was also detected. Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.
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PMID:Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane. 297 65

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100-250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.
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PMID:Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae. 297 29

In this review, the cell envelope of the human pathogenic yeast Candida albicans includes the plasma membrane and the mannoproteins, enzymes, beta-glucans, and chitin of the wall. The organization of the wall is complex and ultrastructural studies show distinct "layers". Mannoprotein is distributed throughout the wall but is concentrated on the exterior surface and adjacent to the plasma membrane. The mannoproteins contain the antigenic determinants of the yeast cells. The major structural components of the wall are beta-1,3- and beta 1,6-glucans, and these two linkages are present in almost equal amounts. Chitin is concentrated at the bud scar, but small amounts are located over the entire wall where it appears to be linked to beta-1,6-glucan. Chemical bonding both within and between wall components confers rigidity on the wall and restricts movement of molecules into and out of the cell. Soluble enzymes are retained within the wall matrix, but a number of enzymes and proteins are excreted. The plasma membrane of C. albicans is similar to that isolated from other fungi and contains the proton pump ATPase and enzymes involved in biosynthesis of the wall such as chitin synthase and beta-1,3-glucan synthase.
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PMID:Cell envelope of Candida albicans. 331 22

Thermal inactivation studies performed with several membrane-bound enzymes such as chitin synthase, cytochrome c oxidase, nucleotidase, as well as bacterial, mitochondrial and plasma membrane ATPases from yeast in H2O and 2H2O indicated that most of these enzymes could not be protected by 2H2O against thermal inactivation. Only the Escherichia coli ATPase, which is located at the surface of the membrane, and the cytochrome c oxidase were stabilized by heavy water.
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PMID:Does 2H2O also protect membrane-bound enzymes? 630 84

The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions.
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PMID:Isolation and characterization of the Neurospora crassa endoplasmic reticulum. 631

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (beta-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.
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PMID:The in situ assay of Candida albicans enzymes during yeast growth and germ-tube formation. 631 58

A method has been developed to isolate plasma membranes with high ATPase activity from wild type Neurospora. Cells are treated with snail enzyme to weaken their cell walls, disrupted by gentle homogenization in a medium designed to keep mitochondria and other organelles intact, and fractionated by differential centrifugation. After removal of mitochondria, several higher speed particulate fractions (particularly one sedimenting at 40,000 X g) contain an ATPase that can be identified as the plasma membrane enzyme on the basis of sensitivity to vanadate and kinetic properties. Its [S]0.5 for Mg.ATP, specificity for nucleotides and divalent cations, and pH optimum are virtually identical with those reported previously for plasma membrane ATPase from the slime mutant of Neurospora (Bowman, B. J., and Slayman, C. W. (1977) J. Biol. Chem. 252, 3357-3363). By contrast, ATPase specific activities in the wild type plasma membranes are much higher than in slime, ranging up to 7.3 mumol/min/mg of protein (the highest value yet reported for Neurospora). The best preparations appear homogeneous upon sucrose density gradient centrifugation, and band at an equilibrium density of 1.15 g/cm3. Two other markers, chitin synthetase and [acetyl-3H] concanavalin A binding, show approximate co-purification with the plasma membrane ATPase through membrane fractionation and sucrose gradient centrifugation.
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PMID:Isolation and characterization of plasma membranes from wild type Neurospora crassa. 645 33

Permeabilization of yeast and other fungal cells by osmotic shock enabled the in situ assays of intracellular plasma membrane-bound enzymes, such as beta-1,3-glucan synthase, chitin synthase, and Na(+)/K(+) ATPase as well as the soluble, cytoplasmic enzymes, such as lactate dehydrogenase and alpha-glucosidase. The permeabilization was accomplished by rapid changes in osmolarity of the washing buffer at 0 degrees C whereby 0.5-3.5 M glycerol, sorbitol, and/or mannitol and/or 1 M KCl could be used as the osmolytes. No appreciable leakage of intracellular proteins occurred during the permeabilization procedure. The described procedure caused practically complete cell permeabilization while avoiding treatments with organic solvents, detergents, and other xenobiotics currently used for the permeabilization of microbial cells.
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PMID:In situ assays of fungal enzymes in cells permeabilized by osmotic shock. 1131 15


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