Metabolism and function of polyphosphates in bacteria and yeast.
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Animals↗
Biomedical subjects
I S Kulaev
Publications and source records attributed to I S Kulaev.
Hydrolysis of tripolyphosphate by purified exopolyphosphatase from Saccharomyces cerevisiae cytosol: kinetic model.
The kinetics of hydrolysis of tripolyphosphate by purified exopolyphosphatase from Saccharomyces cerevisiae cytosol has been studied in the presence of Mg2+. Two kinetic models suggesting the formation of complexes of tripolyphosphate and the enzyme with Mg2+ are compared. Both models suggest that only enzyme--substrate complexes containing Mg2+ and tripolyphosphate simultaneously are able to hydrolyze the tripolyphosphate. The first model suggests that the enzyme is able to bind to Mg2+ independently from substrate binding. The second model does not consider this possibility, but suggests that both complexes containing tripolyphosphate and Mg2+ in proportion 1:1 and 1:2 can serve as the reaction substrates. The description of the experimental data by both models is essentially the same. The complex containing tripolyphosphate and Mg2+ in proportion 1:1 is optimal for the enzyme activity, the complex containing tripolyphosphate and Mg2+ in proportion 1:2 being hydrolyzed at a lower rate.
Acid Anhydride Hydrolases↗
Purification and properties of exopolyphosphatase isolated from Saccharomyces cerevisiae vacuoles.
An exopolyphosphatase (polyPase) with a specific activity of 60 U/mg protein has been purified from the vacuolar sap of Saccharomyces cerevisiae. The molecular mass of the intact enzyme was found to be 245 kDa. It is highly specific towards high-molecular polyphosphates (polyP). The activity with polyP9 is 24% of that with polyP208. The apparent Km for polyP15 and polyP208 hydrolysis is 93 and 2.4 microM, respectively. The enzyme is slightly active with polyP3 and adenosine-5'-tetraphosphate, but does not hydrolyze pyrophosphate, ATP, GTP and p-nitrophenylphosphate. It is stimulated by divalent metal cations. Co2+, the best activator, stimulates it 6-fold. Antibodies that inhibit the cell envelope and cytosol polyPases of S. cerevisiae have no effect on the vacuolar polyPase. The vacuolar polyPase differs from other yeast polyPases in molecular mass, substrate specificity and effects of activators.
Acid Anhydride Hydrolases↗
Purification and characterization of P33 protein of Candida utilis homologous to bgl2p of Saccharomyces cerevisiae; comparative analysis of the role of these proteins in molecular organization of the yeast cell walls.
P33 protein was isolated from the cell walls of Candida utilis. Homology between P33 and Bgl2p proteins from the cell walls of Saccharomyces cerevisiae was shown. The important role of these proteins in molecular organization of yeast cell walls was demonstrated using trypsin proteolysis and the "gene disruption" method.
Candida↗
Comparison of exopolyphosphatases of different yeast cell compartments.
Purified cell-envelope polyphosphatase as well as polyphoshatase activities of cytosol and isolated vacuoles, of nuclei and mitochondria of the yeast Saccharomyces cerevisiae were compared. The polyphosphatases of cell envelope and cytosol are similar, the polyphosphatases of nuclei, vacuoles and mitochondria differ in their kinetic properties, substrate specificity, requirements in divalent cations and in some effector actions both from these and from each other.
Acid Anhydride Hydrolases↗
Transport of phosphate into vacuoles of Saccharomyces cerevisiae.
Isolated vacuoles of the yeast Saccharomyces cerevisiae accumulated orthophosphate at pH 6-7. This accumulation was inhibited by MgATP, and was insensitive to protonophores. Triton X-100 blocked this process. The accumulation increased linear by any phosphate concentrations employed (from 0.2 to 10 mM). It is proposed that phosphate is transported into yeast vacuoles via a channel transport system independent of the electrochemical proton gradient on the vacuolar membrane.
Adenosine Triphosphate↗
Adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate: new substrates of the cytosolic exopolyphosphatase of the yeast Saccharomyces cerevisiae.
A cytosolic preparation of Saccharomyces cerevisiae is capable of hydrolyzing adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate with activities which are 1.5-2 times greater than that with polyP15. The apparent K(m) values for hydrolysis of adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate are 100 and 80 microM, respectively. A comparative study of inhibitors shows that these activities are inherent characteristics of these exopolyphosphatases.
Acid Anhydride Hydrolases↗
Detection and some properties of membrane-bound and soluble polyphosphatases in mitochondria of the yeast Saccharomyces cerevisiae.
Saccharomyces cerevisiae mitochondria possess polyphosphatases that are tightly bound to the membranes and differ from soluble polyphosphatase of these organelles in a number of properties. Molecular weights of the membrane-bound polyphosphatases are 120 and 76 kD, and the molecular weight of the soluble polyphosphatase is about 36 kD. All three enzymes are evidently monomers, since antibodies against purified cell-envelope polyphosphatase of S. cerevisiae reacted with 115, 78, and 37 kD polypeptides in immunoblotting. The activities of membrane-bound and soluble polyphosphatase are maximal at neutral pH. The soluble polyphosphatase activity is stimulated by divalent cations, unlike the membrane-bound enzymes which are inhibited by the same cations including Mg2+. Monovalent cations do not affect the activity of the soluble enzyme but stimulate polyphosphatases in the membrane preparation. The specific activities for hydrolysis of polyphosphates with average chain lengths of 9 to 188 phosphate residues are enhanced by increasing the degree of substrate polymerization in the case of the membrane preparation and are unchanged in case of the soluble enzyme. Affinity of the soluble enzyme to polyphosphates is 5-10 times higher than that of the membrane-bound polyphosphatases. In the soluble fraction of mitochondria, high tripolyphosphatase activity is detected which is approximately 80% of that in isolated mitochondria.
Cations, Divalent↗
[Characteristics of Saccharomyces cerevisiae nuclear polyphosphatase activity].
Saccharomyces cerevisiae nuclei possess a polyphosphatase activity which is insensitive to a number of inhibitors of ATPase and pyrophosphatase (PPase) activities of the same organelle. Heparin, an effective inhibitor of the nuclear polyphosphatase activity, does not alter either the ATPase and PPase activity. The nuclear polyphosphatase activity is optimal at pH 7.5. Bivalent metal cations stimulate this activity in the following order: Co2+ > Mg2+ > Zn2+ > Mn2+. However, the magnitude of the stimulating effect is much lower than that for the polyphosphatase activities from other organelles of the same yeast. The polyphosphatase activity is nearly the same for polyphosphates ranging from [symbol: see text] = 9 to [symbol: see text] = 208, but is 1.5 times higher for tripolyphosphate. The K(m) values for the hydrolysis of polyphosphates with chain lengths [symbol: see text] = 3, 15 and 208 are 100, 5 and 4.1 microM, respectively. The polyphosphatase activity differs in some properties from that of the cell envelope, cytosol and vacuoles of the same S. cerevisiae strain.
Acid Anhydride Hydrolases↗
[Bacteriolytic enzyme preparation lysoamidase. Purification and some properties of bacteriolytic peptidase L1].
The bacteriolytic peptidase L1 has been isolated from the enzyme preparation of lysoamidase capable to lyze cell walls of gram-positive bacteria using ion-exchange chromatography and gel filtration. Some physico-chemical properties of the enzyme have been established. The molecular mass of L1 is 21 kDa, the pH optimum for Staphylococcus aureus cell lysis is 7-11. The optimal concentration of the buffer is 50 mM; the temperature optimum is 70 degrees C; the half-inactivation temperature is 55 degrees C.
Cell Wall↗
[Characteristics of polyphosphatase activity of isolated mitochondria from Saccharomyces cerevisiae].
Saccharomyces cerevisiae mitochondria have a polyphosphatase activity which is insensitive to a number of inhibitors of mitochondrial ATPase and pyrophosphatase (PPase). Heparin (20 micrograms/ml) and EDTA (0.5 mM) do not inhibit ATPase and PPase activities but completely suppressed mitochondrial polyphosphatase activity. The mitochondrial polyphosphatase activity is maximal at neutral pH; it is inhibited by monovalent cations in the presence of Tris+ (K+ > Na+ > NH4+), and stimulated by bivalent metal cations (Co2+ > Mg2+ > Zn2+ > Mn2+). The polyphosphatase activity does not significantly depend on polyphosphate chain length from 9 to 208 but is more than one order of magnitude higher than activity with tripolyphosphate. Some properties of mitochondrial polyphosphatase activity differ from the characteristics of polyphosphatases of cell envelope, cytosol, vacuoles and nuclei of the same S. cerevisiae strain.
Acid Anhydride Hydrolases↗
[Purification and characteristics of Saccharomyces cerevisiae cytosol polyphosphatase].
The polyphosphatase with specific activity of 283 units/mg was purified 3450-fold to homogeneity with 3.8% yield from cytosol of Saccharomyces cerevisiae yeast. Polyphosphatase is monomeric 40 kD protein. The enzyme hydrolyzes polyphosphates of various chain length including tripolyphosphate but ATP, pyrophosphate, and p-nitrophenyl phosphate are not the substrates. Enzyme activity is maximal at 50 degrees C and pH 6.5-8.5. Several cations of bivalent metals stimulated the enzyme activity 8-66-fold (Co2+ > Mn2+ > Mg2+ > Zn2+ > Fe2+). The enzyme is inactive in the presence of Ca2+ or Cu2+. Heparin, antibodies against cell-envelope polyphosphatase, and Cu2+ or Zn2+ in the presence of Mg2+ are potent inhibitors of cytosolic polyphosphatase. Cytosolic polyphosphatase is similar to purified cell-envelope polyphosphatase but differs in some properties from nuclear, vacuolar, and mitochondrial polyphosphatase of the very same yeast.
Acid Anhydride Hydrolases↗
Structure of an acidic polysaccharide present in the bacteriolytic complex lysoamidase.
The structure of an acidic polysaccharide component of a bacteriolytic complex (lysoamidase), isolated from a bacterium of the genus Xanthomonas, was studied. On the basis of sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopic study of the initial polysaccharide and its O-deacetylated and carboxyl-reduced derivatives, the following structure of the trisaccharide repeating unit of the polysaccharide was established [formula: see text] where ManNAcA and GalNAcA are 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2-deoxygalacturonic acid, respectively.
Carbohydrate Conformation↗
[Structure of an acidic polysaccharide found in a bacteriolytic complex of lysoamidase].
A structural study of an acidic polysaccharide, a component of the lysoamidase bacteriolytic complex has been carried out. Analysis of the monosaccharide composition of the original polysaccharide, of the product of carboxyl groups reduction and of the 13C-NMR and 1H-NMR spectra of the original and the O-deacetylated polysaccharides using two-dimensional NMR spectroscopy has made it possible to establish the structure of the repeating trisaccharide unit of the polysaccharide as follows: [formula: see text] where ManNAcA and GalNAcA are 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2-deoxygalacturonic acid, respectively. Also small amount of a neutral polysaccharide containing of rhamnose is present in the lysoamidase preparation.
Bacteria↗
[Organelle specificity of yeast cell polyphosphatases].
The properties of purified cell envelope polyphosphatase, polyphosphatase activities of vacuoles and cytosol fractions of the Saccharomyces cerevisiae yeast have been compared. The whole body of evidence presently available suggest that each of the compartments under study is equipped with its own polyphosphatase which differs from polyphosphatases of other organelles. It is proposed that the organelle specificity of yeast polyphosphatases may reflect the endosymbiotic origin of eucaryotic cells.
Acid Anhydride Hydrolases↗
[Detection of polyphosphatase activity in isolated Saccharomyces cerevisiae nuclei].
Intact nuclei have been isolated from cells of a diploid strain of Saccharomyces cerevisiae. The isolated nuclei were free from cytoplasmic, mitochondrial and vacuolar marker enzymes. The protein to DNA ratio (w/w) was 11. Pyrophosphatase, tripolyphosphatase and exopolyphosphatase activities have been found in S. cerevisiae nuclei for the first time and were equal to 400, 130 and 55 mU/mg of protein, respectively.
Acid Anhydride Hydrolases↗
[Immunoenzyme analysis of polyphosphatases from various compartments of yeast cells].
Antibodies against purified polyphosphatase from the Saccharomyces cerevisiae cell envelope inhibited the activity of this enzyme and the polyphosphatase activity of the cytosol, being without any effect on vacuolar and nuclear polyphosphatase activities from the same yeast species cells. Using immunoblotting, it has been shown that it is the 40 kDa polypeptide that binds to these antibodies in preparations of cell envelope and cytosolic polyphosphatase. The molecular mass of these polyphosphatases determined by other methods was almost indentical. The 72 and 40 kDa polypeptides bind to these antibodies in isolated vacuoles, while the 64 and 32 kDa polypeptides--in isolated nuclei.
Acid Anhydride Hydrolases↗
Purification and characterization of an exopolyphosphatase from Saccharomyces cerevisiae.
An exopolyphosphatase (polyphosphate phosphohydrolase; EC 3.6.1.11) activity that cleaves inorganic polyphosphates to orthophosphate has been purified to apparent homogeneity (> 95% pure) from Saccharomyces cerevisiae. The exopolyphosphatase is a monomeric protein with a polypeptide molecular mass of 28 kDa. The enzyme, which can be stabilized in the presence of Triton X-100, has a pH optimum of 7.5 and requires, for maximal activity, Co2+ or Mg2+ ions. In the absence of these ions, the exopolyphosphatase binds to polyphosphate but does not degrade it, allowing affinity purification of the enzyme on a polyphosphate-modified zirconia support. o-Vanadate, Cu2+, and Ca2+ are effective inhibitors of the exopolyphosphatase. The enzyme preferentially hydrolyzes linear polyphosphates in a non-processive manner; pyrophosphate as well as cyclic tri- and tetrametaphosphate are degraded only at very low rates, whereas ATP is not split by the exopolyphosphatase. The only product formed by the action of the enzyme is orthophosphate.
Acid Anhydride Hydrolases↗