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Changes in orthophosphate, pyrophosphate and long-chain polyphosphate levels in Leishmania major promastigotes incubated with and without glucose.

The intracellular levels of orthophosphate (Pi), pyrophosphate (PPi) and short- and long-chain polyphosphate (Poly P) were measured in Leishmania major promastigotes incubated in a phosphate-free medium. In the absence of exogenous substrate, the levels of both Pi and PPi increased during a 1 h incubation. The increase in both Pi and PPi was prevented when glucose was present, but glycerol prevented the rise in Pi only. A rise in Pi and PPi was also seen in cells incubated in the absence of exogenous substrate under anaerobic conditions. This was reversed upon addition of glucose plus oxygen. Polyphosphate, here shown to be present in L. major, was measured by means of a polyphosphate glucokinase assay. Short-chain Poly P content did not differ between cells incubated for 1 h in the absence of exogenous substrate or in the presence of glucose or glycerol. Long-chain Poly P content, however, was lower in cells incubated without glucose than in cells incubated with glucose and was also lower in cells incubated for 1 h with glycerol as compared with freshly washed cells. Up to 61% of the increase in Pi and PPi that occurred in promastigotes incubated in the absence of exogenous substrate could have arisen from the concomitant decrease in long-chain Poly P.

Animals↗

Polyphosphates in intraradical and extraradical hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita.

The amount of polyphosphate in the intraradical and extraradical hyphae of Gigaspora margarita was estimated from successive extractions with trichloroacetic acid (TCA), EDTA, and phenol-chloroform (PC). In the intraradical hyphae, most of the polyphosphate was present in TCA- and EDTA-soluble (short-chain and long-chain) fractions, whereas most of the polyphosphate in the extraradical hyphae was present in EDTA- and PC-soluble (long-chain and granular) fractions.

Biomass↗

Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli.

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase.

Bacterial Proteins↗

Polyphosphate kinase protects Salmonella enterica from weak organic acid stress.

Mutants of Salmonella enterica lacking polyphosphate kinase (ppk) grow poorly in the presence of the weak organic acids acetate, propionate, and benzoate. This sensitivity is corrected by methionine and seems to result from destabilization of MetA (homoserine transsuccinylase), the first enzyme in methionine biosynthesis. The MetA protein is known to be sensitive to thermal inactivation, and ppk mutants are more sensitive to heat-induced methionine auxotrophy. Peroxide increases the sensitivity of ppk mutants to both heat and acid and may oxidatively damage (carbonylate) destabilized MetA. While acid appears to impair methionine biosynthesis, it leads to derepression of MetA and may inhibit growth by causing toxic accumulation of denatured protein. This is supported by the observation that the overexpression of MetA in ppk mutants causes acid sensitivity that is not corrected by methionine. We propose that polyphosphate acts as a chemical chaperone that helps refold MetA and/or may stimulate proteolysis of toxic denatured protein. The instability of MetA protein may provide a metabolic fuse that blocks growth under conditions that denature proteins; the sensitivity of this fuse is modulated by polyphosphate.

Acids↗

Primary structure of inorganic polyphosphate/ATP-NAD kinase from Micrococcus flavus, and occurrence of substrate inorganic polyphosphate for the enzyme.

The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.

Adenosine Triphosphate↗

Physical enrichment of polyphosphate-accumulating organisms in activated sludge.

Two methods that physically separate polyphosphate-accumulating organisms (PAO) from other organisms in activated sludge were developed. The first method used 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) to selectively stain PAO. When excited with light at 340 nm, polyphosphate granules in DAPI-stained cells fluoresce yellow while cells without polyphosphate fluoresce blue. This difference in fluorescent response was used to separate PAO from non-PAO using flow cytometry. The second method consisted of a simple gradient centrifugation to physically separate PAO from non-PAO based on their density differences. Both methods produced cell suspensions with an increased PAO concentration. From an average PAO concentration of approximately 14% in a full-scale process, the DAPI-flow cytometry method produced sorted samples with PAO representing more than 70% of the total cells, while the density gradient method produced an approximate 43 to 48% PAO enrichment. The physical enrichment methods described herein should facilitate the identification and study of PAO that are relevant in full-scale enhanced biological phosphorus removal processes.

Bacteria↗

Isolation and characterization of a Gram-positive polyphosphate-accumulating bacterium.

A Gram-positive polyphosphate-accumulating bacterium was isolated from phosphate-removal activated sludge using pyruvate-supplemented agar plates. The isolate was oval or coccobacilli (0.4-0.7 x 0.5-1.0 mm) that occurred singly, in pairs or irregular clumps. Polyphosphate granules in the cells were observed by toluidine blue staining. The pure culture of the isolate rapidly took up phosphate (9.2 mg-P/g-dry weight) in the 3-h aerobic incubation without organic substrates, after anaerobic incubation with organic substrates containing casamino acids. When acetate was the sole carbon source in the anaerobic incubation, the isolate did not remove phosphate. These physiological features of the isolate were similar to those of Microlunatus phosphovorus. However, unlike M. phosphovorus the P-removal ability of the isolate was relatively low and was not accelerated by repeating the anaerobic/aerobic incubation cycles. Phylogenetic analysis and comparison of several characteristics showed that the isolate was identified as Tetrasphaera elongata which was recently proposed as a new polyphosphate-accumulating species isolated from activated sludge. As the isolate contained menaquinone (MK)-8(H(4)) as the predominant isoprenoid ubiquinone, it may be significantly responsible for phosphate removal, because MK-8(H(4)) has reportedly been found in fairly high proportions in many phosphate-removing activated sludges.

Gram-Positive Bacteria↗

Availability of phosphorus from ammonium polyphosphate for growing chickens.

Phosphorus from ammonium polyphosphate was equivalent in availability to that of other phosphate sources commonly used in broiler rations, and it was more available than that from reagent-grade tricalcium phosphate. Two three-week experiments conducted with broiler chickens showed that growth rate, feed efficiency, viability, and bone calcification were improved to a similar extent by adding an equivalent increment of phosphorus from dicalcium phosphate, defluorinated phosphate, or ammonium polyphosphate. The results of these experiments suggest that ammonium polyphosphate may be a useful phosphorus supplement for practical poultry rations.

Animals↗

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.

Acid Anhydride Hydrolases↗

Glass transition and enthalpy relaxation of polyphosphate compounds.

The glass transition behaviour of polyphosphate compounds such as di- and tri- polyphosphates used as food additives and ATP, ADP existing in biosystems, were investigated by using DSC. From the DSC heating curves of the frozen solutions, the glass transition temperatures of the maximum freeze concentrated solutions, Tg', were determined. It was found that Tg's for polyphosphates are relatively high. The lyophilized tripolyphosphates, ATP and ADP also showed the glass transition at a relatively high temperature, depending on the moisture content. In addition, the enthalpy relaxation behaviour of glassy ATP and ADP was examined and analyzed by using the Kohlrausch-Williams-Watts (KWW) and the Vogel-Fulcher-Tammann (VFT) equations. Judging from the parameters of the KWW and VFT equations, the amorphous states of ATP and ADP were suggested to be more fragile than trehalose and sucrose.

Adenosine Diphosphate↗

Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration.

Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.

Adenosine Triphosphate↗

[Research advance in polyphosphate-accumulating microorganisms in enhanced biological phosphorus removal process].

This paper discussed the function of enhanced biological phosphorus removal (EBPR) in P pollution control, P containing wastewater treatment and P resources recovery, and summarized the metabolic characteristics, research progress and methodologies of polyphosphate-accumulating organisms (PAOs). Although polyphosphate has been found in many organisms, only few of PAOs were isolated, cultured and identified. Culture medium formulation is the key to isolate PAOs and to study the microbial accumulation of polyphosphate, and the competition of glycogen-accumulating organisms (GAOs) with PAOs for carbon resources is one of the reasons of low EBPR efficiency. Modern scientific methods such as fluorescent in situ hybridization, confocal laser scanning microscope, microautoradiography, and in vivo NMR spectroscopy, provided powerful tools to analyze PAO species composition, spatial structure and functional properties under field conditions. The knowledge of PAO is valuable to enhance the P removal efficiency in water treatment plant, and to improve our understanding on P transformation and transferring in environment.

Bacteria↗

[Peculiarities of metabolism and functions of high-molecular inorganic polyphosphates in yeasts as representatives of lower eukaryotes].

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.

Cytosol↗

[High-molecular polyphosphates and pyrophosphate in cyclosporin- producing Tolypocladium sp. and their role in the processes of growth and antibiotic synthesis].

The contents of high-energy phosphorous compounds, i.e. three fractions of polyphosphates, pyrophosphate, and ATP were determined in isogenic strains of Tolypocladium sp. differing in cyclosporine production levels. The content of polyphosphates was 1 to 2 orders of magnitude greater than that of pyrophosphate or ATP and did not depend on the strain productivity. During the period of the mycelial intensive growth and at the beginning of antibiotic synthesis, the level of polyphosphates lowered 2-3-fold and the content of pyrophosphate markedly decreased as well. The activities of polyphosphatase and pyrophosphatase during the culture growth and cyclosporine biosynthesis was higher in the highly productive strain.

Cyclosporins↗

Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets.

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.

Blood Platelets↗

Hyperthermia induced polyphosphate changes in Propionibacterium acnes as studied by 31P NMR.

The polyphosphate component in 31P NMR spectra of the Gram-positive Propionibacterium acnes increased after hyperthermia treatment. The cells were exposed to temperatures in the interval from 15 degrees C to 45 degrees C. The amount of polyphosphate increased with increasing temperature. There were no temperature induced changes in the other phosphorous components seen in the spectra with exception of a decrease in ATP for higher temperatures. The increase in polyphosphates was less than that obtained from cells irradiated by near ultra-violet light.

Hot Temperature↗

[Stimulating action of polyphosphates on peptide formation from glycine and phenylalanine amides under abiogenic conditions].

Synthesis of peptides during polymerization of GlyNH2 and PheNH2 has been demonstrated by means of gel-chromatography and thin-layer chromatography. The optima of pH and temperature have been estimated for the reaction. Grem's salt, tripolyphosphate and pyrophosphate were shown to cause the stimulatory effect on the peptide formation, but there was no correlation between the yield of the peptides and the hydrolysis of the polyphosphates. On the basis of the experimental data, it has been concluded that hydrolysis of polyphosphates is not an energy source for the polymerization of GlyNH2 and PheNH2. Therefore, polyphosphates cause catalytic effect on the peptide synthesis from the amides of amino acids in homogeneous medium.

Catalysis↗

[Relationship between the content of some fractions of high molecular weight polyphosphates and total nucleic acids upon dehydration of the yeast Saccharomyces cerevisiae].

Essential redistribution of various polyphosphate fractions was shown during dehydration and subsequent reactivation of Saccharomyces cerevisiae 14. Dehydration no matter what method was used, was followed by an increase in the content of acid soluble polyphosphates (fraction Poly P1) and a decrease of that of salt soluble polyphosphates (fraction Poly P2). Reactivation of dehydrated yeast was, on the contrary, accompanied by a decrease in the PP 1 and an increase in the Poly P2 content. A direct correlation between the Poly P2 fraction and total nucleic acids was demonstrated under various conditions of dehydration and subsequent reactivation. An inverse correlation between the content of the Poly P2, fraction and nucleic acids, on the one hand, and that of the Poly P1 fraction, on the other, was observed. Study of activities of polyphosphatases, tripolyphosphatase, pyrophosphatase and ATPase in dehydrated yeast showed values similar to those in original cells.

DNA↗