Inorganic polyphosphates in mitochondria.

Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (approximately 15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7-10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca2+/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting P(i) from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.

[The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum].

The contents of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H(+)-ATPase activity in the plasma membrane were determined in a low-activity cephalosporin C (cephC) producer Acremonium chrysogenum ATCC 11550 and selected highly efficient producer strain 26/8 grown on glucose or a synthetic medium providing for active synthesis of this antibiotic. It was shown that strain 26/8 on the synthetic medium produced 26-fold higher amount of cephC as compared with strain ATCC 11550. This was accompanied by a drastic decrease in the cell contents of ATP and the high-molecular-weight fractions polyP2, polyP3, and polyPS with a concurrent increase in the low-molecular-weight fraction polyP1. These data suggest that polyPs are involved in the cephC synthesis as a source of energy. H(+)-ATPase activity insignificantly changed at both low and high levels of cephC production. This confirms the assumption that A. chrysogenum has other alternative antibiotic transporters in addition to cefT. The obtained results can be used for optimizing commercial-scale cephC biosynthesis.

[Metabolism characteristics of Chelativorans oligotrophicus by two-phase growth on the mixture of EDTA and glucose].

The obligate destructor of ethylene diamine tetraacetate--a culture of Chelativorans oligotrophicus LPM-4--did not grow on a medium with glucose, but it was good to use it under cultivation on a mixture with EDTA after considerable decrease of the EDTA concentration in the medium (two-phase growth). Strong inhibition of hexokinase and glucose 6-phosphate dehydrogenase in cell exracts 4 mM EDTA was revealed. Using EDTA, cells accumulated polyphosphates whose rate decreased during glucose utilization phase. High activities of polyphosphate biosynthesis ferments (adenylat kinase and polyphosphate kinase) were distinguished during the first phase of the cultivation; considerable decrease of them and increase of polyphosphate glucokinase were found during the second phase of the cultivation. This points to the possible participating of polyphosphates in glucose metabolism as a supplementary energy source.

[Study of the content of inorganic polyphosphates in Saccharomyces cerevisiae grown on different carbon sources with different O2 concentrations in the medium].

The content of different fractions of inorganic polyphosphates (polyP) was studied in Saccharomyces cerevisiae VKM Y-1173 growing on a complete medium with glucose under hypoxia and active aeration as well as on ethanol. The highest growth rate was observed for aerobic fermentation, while the yield of biomass was maximal for cultivation on ethanol. In the mid-log growth phase, the amount of polyP was maximal in the cells grown on glucose under hypoxia and minimal on ethanol. In this latter case, the content of different polyP fractions changed unevenly: polyP3, polyP4, and polyP1 decreased by approximately 60%, 45%, and 30%, respectively; the salt-soluble polyP2 remained at almost the same level; while polyP5 abruptly increased 10- to 15-fold. These findings demonstrate that the metabolic pathways for polyP fractions are different. A significant drop in the amount of the main polyP fractions accompanied by a decrease of the polyP average chain length in the presence of carbon and Pi sources in the medium is evidence of active involvement of polyP as additional energy sources in the flows of energy in actively growing yeast cells.

[Effects of cellobiose lipid B on Saccharomyces cerevisiae cells: K+ leakage and inhibition of polyphosphate accumulation].

Cellobiose lipid B, a natural fungicide produced by the yeast Pseudozyma fusiformata, induces the leakage of K+ and ATP from cells of Saccharomyces cerevisiae. The presence of glucose decreases the effective concentration of cellobiose lipid B. The concentration of cellobiose lipid B was selected that results in a high rate of K+ leakage and a five- to sevenfold decrease in the intracellular ATP content, while the accumulation of acid-soluble polyphosphates decreased only by half. These results indicate the possibility of synthesis of these polymers independently of the ATP level and of the ion gradient on the plasma membrane.

Efflux of potassium ions from cells and spheroplasts of Saccharomyces cerevisiae yeast treated with silver and copper ions.

Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-beta-D-glucopyranosyl-(1-->4)-(6-O-acetyl-beta-D-glucopyranosyl-(1-->16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H+-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K+ transport systems during preparation of spheroplasts.

[The effect of inactivation of the exo- and endopolyphosphatase genes PPX1 and PPN1 on the level of different polyphosphates in the yeast Saccharomyces cerevisiae].

The inactivation of the PPX1 and PPN1 genes, which encode the major enzymes of polyphosphate degradation (exopolyphosphatase and endopolyphosphatase, respectively), was found to exert different effects on the content of different polyphosphates in the yeast Saccharomyces cerevisiae. The content of relatively low-molecular-weight acid-soluble polyphosphates in mutant yeast strains is inversely proportional to the exopolyphosphatase activity of the cytosol. At the same time, the mutation of these genes exerts no effect on salt-soluble polyphosphates. The content of high-molecular-weight alkali-soluble polyphosphates increases twofold in a mutant with inactivated genes of both exopolyphosphatase and endopolyphosphatase. The data obtained confirm the earlier suggestion that the metabolic pathways of particular polyphosphates in yeasts are different.

Accumulation of polyphosphates and expression of high molecular weight exopolyphosphatase in the yeast Saccharomyces cerevisiae.

The effect of cultivation time and concentration of inorganic phosphate (P(i)) in the culture medium on the accumulation of polyphosphates (polyP) and the activity of two cytosolic exopolyphosphatases of the yeast Saccharomyces cerevisiae was studied: an exopolyphosphatase of 40 kD encoded by PPX1 and a high molecular weight exopolyphosphatase encoded by another gene. Depletion of polyP in the cells on P(i) starvation is a signal factor for the accumulation of polyP after the subsequent addition of 5-20 mM P(i) and glucose to the cells or spheroplasts. A high activity of both exopolyphosphatases does not prevent the accumulation of polyP. The expression of the high molecular weight exopolyphosphatase is due to the acceleration of metabolism in cells that have reached the stage of growth deceleration on the addition of P(i) and glucose or complete culture medium. This process may occur independently from the accumulation of polyP. The activity of exopolyphosphatase PPX1 depends less on the mentioned factors, decreasing 10-fold only under conditions of phosphate surplus at the stationary growth stage.

Effect of inhibitors on polyphosphate metabolism in the yeast Saccharomyces cerevisiae under hypercompensation conditions.

After re-inoculation of the yeast Saccharomyces cerevisiae from phosphate-deficient to complete medium, the total content of polyphosphates increased tenfold during 2 h (hypercompensation), but the content of certain fractions increased differently. The content of acid-soluble polyphosphate increased to the maximal extent. The ratio of the activities of two exopolyphosphatases also changed in the cytosol. Activity of a low molecular weight exopolyphosphatase (40 kD) decreased almost twice, whereas activity of a high molecular weight exopolyphosphatase (830 kD) increased tenfold. Cycloheximide blocks the increase in activity of high molecular weight exopolyphosphatase and hence, under these conditions the latter is synthesized de novo. Inhibitors of energy metabolism and cycloheximide, an inhibitor of protein synthesis, differently influence accumulation of certain polyphosphate fractions under hypercompensation conditions. The effect of iodoacetamide, an inhibitor of glycolysis, on any fraction is negligible, while cycloheximide suppresses accumulation of only polyP4 fraction associated with the cell envelope and bafilomycin A1, an inhibitor of vacuolar H+-ATPase, suppresses accumulation of polyP3 fraction. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to variable extent inhibits accumulation of all the fractions. Analysis of the effect of inhibitors on accumulation of polyphosphates under hypercompensation conditions confirms various localization, heterogeneity, and multiplicity of the routes of biosynthesis of certain fractions of these macroergic phosphorus compounds and also suggests interrelation between their biosynthesis and the gradient of H+ electrochemical potential.

The content and chain length of polyphosphates from vacuoles of Saccharomyces cerevisiae VKM Y-1173.

The content of inorganic polyphosphates (polyP) in vacuoles of the yeast Saccharomyces cerevisiae is approximately 15% of the total cellular polyP. Over 80% of the vacuole polyP are in an acid-soluble fraction. It was first established by (31)P-NMR spectroscopy that a polymeric degree (n) of two subfractions obtained by precipitation with Ba(2+) in succession at pH 4.5 and 8.2 was approximately 20 +/- 5 and 5 +/- 2 residues of ortho-phosphoric acid, respectively. Under a deficit of phosphate (P(i)) in the cultivation medium, the polyP content in vacuoles decreased approximately 7-fold with the same drastic reduction of their content in the cell. Unlike intact yeast cells, where polyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect. The data indicate the occurrence of special regulatory mechanisms of polyP synthesis in vacuoles differing from those in the whole cell.

The development of A. N. Belozersky's ideas in polyphosphate biochemistry.

This review covers some trends and approaches to the study of inorganic polyphosphates that originated from the fruitful ideas and pioneering works of A. N. Belozersky. This is, first of all, the elucidation of a close relationship between these biopolymers and nucleic acids in organisms at different evolutionary stages; second, the study of "fossil" reactions in polyphosphate metabolism that permit an understanding of their role in the evolution of phosphorus turnover and cell bioenergetics; third, the possible use of the conservative enzymes of polyphosphate metabolism, e.g., exopolyphosphatases, as molecular chronometers for obtaining additional data concerning the theory of the endosymbiotic origin of eukaryotic cells from prokaryotes.

Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae.

The content of inorganic linear polyphosphate (polyP) and the polymeric degree (n) of these compounds were determined in the process of growth of the yeast Saccharomyces cerevisiae VKM Y-1173 in a medium, which contained varying Pi amount with the constant level of all the necessary components. For this purpose, a combination of chemical methods of polyP extraction and 31P-NMR spectroscopy studies of their chain length were used. After 7 h of phosphate starvation, the yeast was shown to use almost completely the phosphate reserve in the form of polyP localized in various cell compartments to support their vitality. The polyP drop was followed by a considerable shortening of the polymer chain length of acid-soluble (polyP1) and two alkali-soluble (polyP3 and polyP4) fractions. Under the same conditions, the content of a salt-soluble fraction (polyP2) decreased almost 20-fold followed by a simultaneous increase of the chain length nearly 2-fold. As a result, fraction chain length ranged up to n = 40-45. Replacement of the yeast cells after phosphate starvation to a complete phosphate- and glucose-containing medium resulted in super-accumulation ("overcompensation") of polyP within 2 h mainly in polyP3 and, to a lesser degree, in polyP1, polyP2, and polyP5 fractions. In polyP4 fraction localized as polyP3 at the cell surface, the polyP super-accumulation was not detected. The increase of polyP amount in the fractions mentioned turned out not to be accompanied by simultaneous elongation of their chain length and occurred at the lowest level that is characteristic of a polymer level for each fraction. Further cultivation of the yeast on the complete medium during 2 h had little or no effect on polyP content in the cells but led to elongation of polyP chain length especially in the polyP3 and polyP4 fractions. A phenomenon of considerable elongation of polyP chain length against the background of their fixed content revealed in the yeast growing on the complete medium suggests that these organisms possess a previously unknown discrete way of polyP biosynthesis, which results first in the formation of comparatively low-molecular-mass chains followed by that of high-molecular-mass polymers.

[Effect of conditions od Trichosporon pullulans culturing on the synthesis of immunomodulating glycoprotein]. / Vliianie usloviu kul'tivirovaniia Trichosporon pullulans na sintez glikoproteina-immunomoduliatora.

An extracellular glycoprotein (GP) exhibiting immunomodulating activity produced by the yeast Trichosporon pullulans grown in a defined ethanol-containing medium differed substantially in its composition from that of the yeast cell walls: therefore, it cannot be considered a structural component of the cell walls. In batch culture, the greatest GP production (40 mg/l) occurred in the exponential phase of the yeast growth. Under continuous cultivation, in both chemostat and pH-auxostat regimes, the specific rate of GP synthesis (qGP) increased with the increasing specific growth rate (mu) and reached 1.55 mg/(g h) at mumax. Under limitation of the yeast growth by zinc qGP was three times lower than under nitrogen or iron limitation. The rate of GP production depended inversely on the oxygen concentration.

[Hypochromic effect of signal attenuation in 31P-NMR spectra of linear polyphosphates]. / Gipokhromnyi éffekt snizheniia intensivnosti signalov v 31P-IaMR spektrakh lineinykh polifosfatov.

Chemical shifts in 31P-NMR spectra of linear polyphosphates were studied. In each polyphosphate species tested, the sum of signal intensities of the internal (core) phosphate groups was proportional to the concentration of each polyphosphate, but the contribution of such groups to the total intensity of the signal decreased with increasing the length of the polyphosphate chain. An equation for estimating the polyphosphate chain length in biological objects taking into account a decrease in the 31P-NMR spectral intensity is proposed.

[Change in inorganic polyphosphate chain length depending on the stage of Saccharomyces cerevisiae growth]. / Izmenenie dliny tsepi neorganicheskikh polifosfatov v zavisimosti ot stadii rosta Saccharomyces cerevisiae.

The dynamics of the content and the degree of polymerization of polyphosphates (PPs) during the growth of Saccharomyces cerevisiae VKM Y-1176 was studied by chemical methods and P NMR spectroscopy. The overall PP content of Saccharomyces cerevisiae cells growing in orthophosphate-sufficient medium was found to increase until glucose was exhausted in the medium. In the early logarithimic phase, the degree of polymerization of PPs (high-polymeric PPs in particular) fell drastically. Further cultivation of yeast in the glucose-depleted medium led to a decrease in the cellular content of all PP fractions without any considerable shortening of their chain length; by the 24th h, high-polymeric PPs accumulated in the cells. Shortening of PPs against a background of their intense synthesis in yeast cells growing in glucose-and orthophosphate-sufficient medium can be explained by the involvement of so far unknown mechanisms of activation of polyphosphate-depolymerizing enzymes.

[Properties of polyphosphate phosphohydrolase of the "leaky" mutant Neurospora crassa with respect to the specific enzyme]. / Izuchenie svoistv polifosfatfosfogidrolazy "leaky" mutanta Neurospora crassa po dannomu fermentu.

Some properties of polyphosphate phosphohydrolase from N. crassa strain ad-6.28610a and from its mutant with a decreased polyphosphate phosphohydrolase activity were compared. It was shown that the pH optimum for both enzyme species lies within the range of 7.1-7.3; the temperature optimum is 45 degrees. The mutant polyphosphate phosphohydrolase has a V value, which is 2 times less than that of the parent strain, and possesses a higher thermal inactivation stability. The enzymes of both cultures have practically identical values of Km(app), which depend on the length of the substrate chain. Upon transition from polyphosphate n = 9 to polyphosphate n = 180 the enzyme affinity is increased. Electrophoretic separation of cell-free extract proteins in polyacrylamide gel revealed the existence of two enzyme isoforms in both strains. The electrophoretic mobility of these isoforms are identical in both cultures. Biosynthesis of polyphosphate phosphohydrolase and tripolyphosphatase of N. crassa, unlike that of bacteria, is not controlled by a system common for alkaline phosphatase.