Search

The review presents the recent data demonstrating the important role high-molecular inorganic polyphosphates in regulatory processes in a yeast cell. It has been shown that polyphosphates are localized in different cell compartments, where they are metabolized by a special set of enzymes. The review presents the evidence in favor of the concept of multiple functions of these biopolymers in a cell, as well as the data on the pleiotropic effects of mutations in the genes encoding the enzymes of polyphosphate metabolism.

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 mumol Pi/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content of H. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities of H. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase of H. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective Km of this enzyme with respect to pyrophosphate was 115 microM.

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.

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.

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.

The cytosol fraction purified from cellular organelles was obtained from S. cerevisiae yeast cells. This cytosolic fraction contained a polyphosphatase activity comprising nearly 65% of such in the protoplast homogenate. The pH optimum of this activity was 6.5-7.5. Bivalent metal cations stimulated the polyphosphatase activity 9-14-fold in the following order: Zn2+ > Co2+ > Mg2+, Mn2+. Fe2+, Cu2+ and Ca2+ ions inhibited this activity at all concentrations used. The cytosolic polyphosphatase was effectively inhibited by molybdate and heparin. Heparin was effective only in the presence of Mg2+, Mn2+ and Co2+ but had no effect in the presence of Zn2+. Heparin inhibited polyphosphatase competitively. The polyphosphatase activity was the same with poly (P)9-poly (P)208. On poly (P)9, poly (P)15 and poly (P)208 the Km(app) values were equal to 19, 11, 1.2 microM, respectively. The molecular mass of cytosolic polyphosphatase determined by gel filtration on Sephacryl S-300 was 50 kDa.

Vacuoles of the Saccharomyces cerevisiae yeast possess a polyphosphatase activity which differs from other known vacuolar phosphohydrolase activities by pH-optimum, sensitivity towards inhibitors and distribution between the tonoplast and vacuolar sap. The polyphosphatase activity is inhibited by EDTA, molybdate, ortho-vanadate and fluoride. Nearly 77% of this activity is located in the vacuolar sap, while 25%--in the tonoplast fraction. Both the soluble and membrane-bound polyphosphatase activities are maximal at pH 6.8-7.2. Bivalent metal cations stimulate this activity in the following order: Zn2+ > Mg2+ > Co2+ > Mn2+. Other ions (Fe2+, Cu2+, Ca2+) inhibit this activity at all concentrations tested. The polyphosphatase activity of both the vacuolar sap and the tonoplast increases 2.4-and 2-fold, respectively, with an increase in the degree of substrate polymerization--from n = 3 to n = 208.

From the culture liquid filtrate of Actinomyces janthinus 118 a trypsin inhibitor (TI-AjI) was isolated by sulphate ammonium precipitation, DEAE-cellulose chromatography, QAE-Sephadex chromatography and two-fold crystallization. The resultant preparation was homogeneous during polyacrylamide gel electrophoresis and ultracentrifugation. As measured by gel filtration, the TI-AjI molecular weight was estimated to be 13,000+/-1,000. The culture liquid of Act. janthinus 118 was found to contain another substance with anti-trypsin activity that differed from TI-AjI in its molecular weight.

The influence of mass exchange and cultivation temperature on the synthesis of trypsin inhibitor in the submerged culture of Actinomyces janthinus was studied. Mass exchange parameters of the fermenter varied from 0.72 to 3.7 g O2 l/hr without oxygen limitations, and cultivation temperature ranged from 25 to 34 degrees C. The growth pattern, dynamics of substrate consumption and synthesis of trypsin inhibitor by the culture of Act. janthinus were shown to depend on mass exchange and cultivation temperature. With an increase in mass exchange the inhibitory activity reached maximum earlier but did not rise in its absolute value. With a temperature increase the inhibitory activity grew by 65%.

The effect of methylation antagonists (DL-ethionine, S-adenosyl homocysteine and folic acid analogs-aminopterine, ametopterine and tomisine) on vitamin B12 and porphyrin biosynthesis of P. shermanii resting cells was studied. DL-ethionine proved the most active inhibitor. Inhibition of vitamin B12 biosynthesis induced by DL-ethionine was accompanied by increase in porphyrin formation. DL-methionine reversed the inhibitory effect of DL-ethionine on the vitamin synthesis. Other compounds tested showed a less marked effect.

Some properties of protein inhibitor for trypsin (TI) from Act. janthinus 118 were studied. It was shown that TI has an antitrypsin activity within a wide pH range with a maximum at about 9,5. At 4 degrees and 20 degrees C TI is stable for 24 hours within the pH range of 6,0--11,0. At 100 degrees C TI is more stable in the slightly acid region of pH than at neutral or alkaline conditions. Trypsin and chymotrypsin inactivate the inhibitor for 8 hours. TI inhibits trypsin, fibrinolysin, subtilisin, pronase and terrilytin, but have no effect on chymotrypsin, thrombin, papain and pepsin. The dissociation constants for the trypsin-inhibitor complex were found to be 1,7.10-8 M, 4,1.10-9 M and 2,4.10-10 M, with casein, p-nitroanilide benzoylarginine and tosylarginine methyl ester used as substrates, respectively. The corresponding dissociation rate constants for the subtilisin-inhibitor complex were equal to 1.10-9 M and 4.10-10 M with casein and carbobenzoxy-L-alanyl-L-alanyl-L-leucin p-nitroanilide used as substrates, respectively.

The inhibiting activity of filtrates of the cultural broth against trypsin and chymotrypsin was studied among 66 actinomycetes. The highest activity against trypsin was found, after selection, in the following cultures: Act. janthinus 118, Act. violatus 125, Act. violaceus confinus 2476, Act. violaceus vicinus 1074. The antitrypsin activity was detected in the cultural broth of Act. janthinus 118 during the first day of its growth, and reached maximum by the third day. The inhibiting substance in the cultural broth is thermo- and pH-stable, is not extracted with organic solvents, and remains in the bag during dialysis. Apparently, the inhibitor (s) of trypsin produced by Act. janthinus 118 differes from trypsin inhibitors of microbial origin and low molecular weight which have been described so far.

The amino acid composition of the protease (TI-Ajl) inhibitor from Actinomyces janthinus 118 has been determined. It was shown that the TI-Ajl, S-SI and plasminostreptin inhibitors of trypsin and subtilisin have a number of common features: number of double bonds, tryptophane and tyrosine residues, prevalence of the acidic amino acids over the basic ones, ets. The effect of chemical modification of amino groups, arginine, tyrosine and methionine residues on the inhibitory activity of TI-Ajl was studied. The data obtained are indicative of the presence of two active centers of the inhibitor. The antitrypsin center contains a lysine residue.