The possible role of high molecular weight polyphosphates in chlortetracycline biosynthesis by Streptomyces aureofaciens]

The content of condensed inorganic polyphosphates in high productive and low productive Streptomyces aureofaciens strains has been determined. At all the stages of growing the quantity of these compounds in low productive strain is 8-10-fold higher than in high productive strains. Maximum accumulation of condensed inorganic polyphosphates in both the strains Str. aureofaciens corresponds to the end of logarithmic phase of growth. In both strains the presence of two enzymes of polyphosphate biosynthesis -- ATP:polyphosphate-phosphotransferase and 1.3-diphosphoglycerate:polyphosphate-phosphotransferase has been revealed as well as the presence of the enzymes of their utilisation -- polyphosphate:D-glucose-6-phosphotransferase, polyphosphate-phosphohydrolase, tripolyphosphosphate-phosphohydrolase and pyrophosphate-phosphohydrolase. The difference in the activity of 1.3 diphosphoglycerate: polyphosphate-phosphotransferase and polyphosphate-phosphohydrolase has been revealed in both strains studied. On the basis of the data obtained it is supposed that there is a possibility of using phosphorus and energy of high molecular inorganic polyphosphate in the biosynthesis of so called products of "secondary metabolism".     

Inorganic polyphosphate metabolism in Staphylococcus aureus and the action on it of antibiotics]

The culure of Staph. aureus in the exponential growth phase contained 14-18 mg/g of dry orthophosphate biomass and 18-22 mg/g of dry acid insoluble polyphosphate biomass. The extracellular extract of the culture had a phosphohydrolase activity with respect to high molecular polyphosphates, tripolyphosphate and pyrophosphate. Penicillin and bacitracin which inhibited the biosynthesis of the cell wall had no effect on the content of polyphosphates and the phosphohydrolase activity of Staph. aureus. Heliomycin which inhibited the biosynthesis of RNA increased the content of polyphosphates by 1.5 times and decreased the content of ATP by 30 per cent in the cells of Staph. aureus.     

Effect of inorganic phosphate on levorin biosynthesis and on the mycelial makeup of Streptomyces levoris]

The effect of inorganic phosphate on biosynthesis of the polyenic antibiotic levorin by Streptomyces levoris and composition of the culture mycelium was studied. It was found that the synthetic medium with 0.4 mM of phosphate was optimal for growth of Str. levoris. When the concentration of phosphate was higher, the biomass increased, while the synthesis of levorin appeared to be inhibited and morphological changes in the culture were observed. Phosphate had a significant effect on the mycelium composition. When its concentration was increased 10 times as compared to the optimal one, the amounts of protein, RNA, total phosphorus and polyphosphates increased 1.3--1.4, 1.6--1.7, 2--3 and 10 times respectively, while the synthesis of levorin decreased 5 times. Changes in the lipid component of the mycelium were also observed. In the absence of inorganic phosphate in the medium the acetone precipitating fraction of the lipids contained 20--40 per cent of the phosphoruless compounds. During cultivation their portion increased up to 70--77 per cent. However, in the presence of its excess the polar lipids were represented only by phospholipids during the whole life cycle. The fatty acid spectrum of the lipids did not depend on the phosphate concentration and was represented mainly by saturated fatty acids with a branched chain of a series of iso- and anteiso-structures containing 14--18 carbon atoms.     

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.     

Effects of inactivation of the PPN1 gene on exopolyphosphatases, inorganic polyphosphates and function of mitochondria in the yeast Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae with inactivated endopolyphosphatase gene PPN1 did not grow on lactate and ethanol, and stopped growth on glucose earlier than the parent strain. Their mitochondria were defective in respiration functions and in metabolism of inorganic polyphosphates. The PPN1 mutants lacked exopolyphosphatase activity and possessed a double level of inorganic polyphosphates in mitochondria. The average chain length of mitochondrial polyphosphates at the stationary growth stage on glucose was about 15-20 and about 130-180 phosphate residues in the parent strain and PPN1 mutants, respectively. Inactivation of the PPX1 gene encoding exopolyphosphatase had no effect on respiration functions and on polyphosphate level and chain length in mitochondria.     

Metabolism characteristics of <Emphasis Type="Italic">Chelativorans oligotrophicus</Emphasis> 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.     

Preferential utilization of inorganic polyphosphate over other bioavailable phosphorus sources by the model diatoms Thalassiosira spp.

Polyphosphates and phosphomonoesters are dominant components of marine dissolved organic phosphorus (DOP). Collectively, DOP represents an important nutritional phosphorus (P) source for phytoplankton growth in the ocean, but the contribution of specific DOP sources to microbial community P demand is not fully understood. In a prior study, it was reported that inorganic polyphosphate was not bioavailable to the model diatoms Thalassiosira weissflogii and Thalassiosira pseudonana. However, in this study, we show that the previous finding was a misinterpretation based on a technical artefact of media preparation and that inorganic polyphosphate is actually widely bioavailable to Thalassiosira spp. In fact, orthophosphate, inorganic tripolyphosphate (3polyP), adenosine triphosphate (ATP) and adenosine monophosphate supported equivalent growth rates and final growth yields within each of four strains of Thalassiosira spp. However, enzyme activity assays revealed in all cultures that cell-associated hydrolysis rates of 3polyP were typically more than ~10-fold higher than degradation of ATP and the model phosphomonoester compound 4-methylumbelliferyl phosphate. These results build on prior work, which showed the preferential utilization of polyphosphates in the cell-free exudates of Thalassiosira spp., and suggest that inorganic polyphosphates may be a key bioavailable source of P for marine phytoplankton.     

Nuclear polyphosphate as a possible source of energy during the sporulation of Physarum polycephalum

31P NMR spectroscopic analysis of the polyphosphate pool in cellular and nuclear extracts of Physarum polycephalum demonstrates that plasmodia and cysts contain inorganic polyphosphates with an average chain length of about 100 phosphates. However, only during sporulation are these high-molecular-weight polyphosphates degraded to a lower molecular weight corresponding to an average chain length of about 10 phosphates. Since polyphosphates are degraded even in the presence of a sufficiently large pool of inorganic phosphate, produced by intracellular injection, we conclude that the degradation of polyphosphates serves in supplying energy for biosynthesis during sporulation rather than in increasing the availability of phosphate.     

Regulation of ANP-stimulated guanylate cyclase in the presence of Mn2+ in rat lung membranes

The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn²⁺ or Mg²⁺. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg²⁺. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn²⁺ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn²⁺ than in the presence of Mg²⁺. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn²⁺ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn²⁺.     

Possible role of high molecular weight polyphosphates in ATP synthesis from exogenous adenine by the culture of Corynebacterium sp., strain VSTI-301]

An addition of exogenous adenine to an autolysing 72-hour culture of Corynebacterium sp., strain BSTI-301 results in accumulation of as much as 0,6--1,0 mp of ATP per 1 ml of medium. Extracellular ATP accumulation under such conditions is coupled with a considerable decrease of the intracellular content of 5'-phosphoribosyl-1-pyrophosphate, orthophosphate, pyrophosphate and two fractions of high-polymeric polyphosphates PPh3 and PPh4, as compared to the control. The activity of pyrophosphate phosphohydrolase (EC 3.6.1.1) and polyphosphate phosphohydrolase (EC 3.6.1.11) is thereby considerably decreased in the cells growing on exogenous adenine, while the activity of ADP-phosphotransferase (EC 2.7.4.1) is increased 2-fold. It was found that in experiments with 14C-adenine the intracellular content of both ATP and ADP remains unchanged despite a considerable accumulation of extracellular ATP in Corynebacterium sp., strain BSTI-301 cells.     

Inorganic polyphosphates and sensitivity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells to membrane-damaging agents

The leakage of ATP and potassium ions from the cells of Saccharomyces cerevisiae with different levels of inorganic polyphosphate was studied under the action of two detergents (natural cellobiose lipid 16-[6-O-acetyl-2′-O-(3-hydroxyhexanoyl)-β-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid and sodium dodecyl sulfate) and silver cations. Cellobiose lipid had practically the same membrane-damaging activity against the cells grown in phosphate-containing medium, under phosphate starvation, and under polyphosphate hypercompensation. The cells grown under the latter conditions were less sensitive to sodium dodecyl sulfate and silver cations. The possible protective action of polyphosphates against the membrane-damaging agents under study is discussed.     

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co²⁺ and Mg²⁺, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co²⁺ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg²⁺ was activated by ADP and arginine but inhibited by ATP.     

Polyphosphate as a source of phosphoryl group in protein modification in the archaebacterium Sulfolobus acidocaldarius

The incubation of polyphosphates with the ribosomal fraction of Sulfolobus acidocaldarius leads to the covalent attachment of phosphate to threonine residue(s) of a single 40,000 Mr protein. The hydrolysis kinetics of this protein showed that polyphosphate might be the modifying group. The reaction requires 2 mM Mn2+ ions and is time-dependent. ATP strongly inhibits the transfer of phosphate from polyphosphate, indicating that this process is catalyzed by an enzyme differing from the well-known protein kinases.     

Inorganic Polyphosphate and Cancer

This review presents data on the relationship between inorganic polyphosphate metabolism and carcinogenesis including participation of polyphosphates in the regulation of activity of mTOR and other proteins involved in carcinogenesis, the role of h-prune protein (human polyphosphatase) in cell migration and metastasis formation, the prospects for using polyphosphates and inhibitors of polyphosphate metabolism enzymes as agents for controlling cell proliferation and migration.     

Some pathways of biosynthesis and degradation of polyphosphates from green algae Acetabularia mediterranea]

The activity of ATP: polyphosphate phosphotransferase was detected in free-cellular extracts of Acetabularia mediterranea. The enzyme activity in cells originally deficient in phosphorus and subsequently transferred into the phosphate-containing medium increases 5-10-fold as compared to normal. Polyphosphate degradation in A. mediterranea is probably produced by polyphosphatase, which was also detected in the free-cellular extract. It was shown that the polyphosphatase activity has two pH optima, i.e. 4.5 and 7.5, and is considerably increased when the cells are transferred into the phosphate-free medium. It is assumed that high-molecular polyphosphates involved in A. Mediterranea metabolism are responsible for regulation of orthophosphate and ATP level in the cells by ATP: polyphosphate phosphotransferase and polyphosphatase.     

Phosphate dependency of phosphofructokinase 2

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP. Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.     

Polyphosphate kinase from Propionibacterium shermanii: formation of an enzymatically active insoluble complex with basic proteins and characterization of synthesized polyphosphate

Polyphosphate kinase, which catalyzes the synthesis of polyphosphate from ATP, has been partially purified from Propionibacterium shermanii. The reaction is unusual in that addition of basic protein causes the enzyme to precipitate and the insoluble form has optimal activity. The synthesized [32P]polyphosphate is non-covalently bound to the precipitated material and was isolated from the complex by proteolysis. The gel electrophoresis procedure of Maxam and Gilbert was adapted to sizing polyphosphates. When polyphosphate was treated with alkali, polyphosphates ranging from 1-100 phosphate residues were obtained as individual bands. The untreated enzymatically synthesized polyphosphate migrated as a species in excess of 200 phosphate moieties.     

D-Fructose 2,6-bisphosphate: a naturally occurring activator for inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase in plants

Inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from mung beans ($\text{Phaseolus}\text{aureus}\text{Roxb.}$) was activated markedly by D-fructose 2,6-bisphosphate, with a K_A of about 50 nM. The enzyme exhibited hyperbolic kinetics both in the absence and presence of the activator. D-Fructose 2,6-bisphosphate (1 μM) decreased the K_m for D-fructose 6-phosphate 67-fold (from 20 mM to 0.3 mM) and increased the V_max 15-fold; these two effects combined to give a 500-fold activation at 0.3 mM D-fructose 6-phosphate. In contrast, ATP:D-fructose 6-phosphate 1-phosphotransferase from the same source was found not to be affected by D-fructose 2,6-bisphosphate.A natural activator for inorganic pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase was isolated from mung-bean extracts and identified as D-fructose 2,6-bisphosphate.     

Basic amino acids and inorganic polyphosphates in Neurospora crassa: independent regulation of vacuolar pools

At least 78%, and perhaps all, of inorganic polyphosphate is shown to be contained within the vesicles (vacuoles) of Neurospora crassa, where over 97% of the soluble arginine, lysine, and ornithine pools are known to accumulate. Furthermore, synthetic polyphosphate can concentrate arginine up to 400-fold from dilute (0.01 mM) solutions in equilibrium dialysis. For these reasons and because the molar ratio of basic amino acids and polyphosphate phosphorus is approximately 1, we tested the hypothesis that there was an obligate physiological relationship between them. Experiments in which nitrogen starvation and arginine excess were imposed upon cells showed that polyphosphate content was insensitive to changes in the basic amino acid content. Experiments involving phosphate starvation and restoration showed that basic amino acid content was almost wholly independent of polyphosphate pools. Moreover, the normal high degree of compartmentation of arginine in vesicles was maintained despite polyphosphate depletion, and arginine was still exchanged across the vesicular membrane. We conclude that N. crassa, like yeasts, can regulate polyphosphates and basic amino acids independently, and that the accumulation of basic amino acids in vesicles may depend upon an energy-requiring mechanism in addition to the demonstrated charge interaction with polyphosphate.