High Molecular Weight Polyphosphates Research Articles

Experimental models of pulmonary embolism

Pulmonary embolism (PE) ranks third in the structure of acute cardiovascular diseases. Every year there is a rapid increase in morbidity and mortality from PE. Laboratory biomarkers for PE diagnosis do not have the necessary specificity, and therefore are ineffective. PE requires timely active treatment, in particular for the prevention of serious complications. In this regard, further research is needed to study and search for novel promising biomarkers for the early detection of PE, pathophysiological mechanisms and targets for therapeutic effects. To a large extent, novel data on the pathophysiology of cardiovascular diseases, including PE, scientists receive from experimental studies using animal models. In this review, we summarize the main existing experimental models of PE, describe the principles and methods for modeling this disease. There are following models of PE: intravenous thrombin infusion, adenosine diphosphate-induced PE, PE induction by thromboplastin, recombinant human tissue factor or high molecular weight polyphosphates, collagen/adrenaline-induced PE, ex vivo thrombus intravenous administration, surgical model. This publication also presents our own experience in creating an artificial model of PE in animals using an intravenous thrombus. In our model, confirmation of PE was obtained during pathological examination and an increase in the level of following biomarkers: troponin, N-terminal pro-brain natriuretic peptide, and D-dimer. In this pilot study, a PE model was created to study the pathogenesis and novel treatment options for this disease. To confirm the effectiveness of the model, future studies are required.

Horizontal transfer of bacterial polyphosphate kinases to eukaryotes: implications for the ice age and land colonisation

BackgroundStudies of online database(s) showed that convincing examples of eukaryote PPKs derived from bacteria type PPK1 and PPK2 enzymes are rare and currently confined to a few simple eukaryotes. These enzymes probably represent several separate horizontal transfer events. Retention of such sequences may be an advantage for tolerance to stresses such as desiccation or nutrient depletion for simple eukaryotes that lack more sophisticated adaptations available to multicellular organisms. We propose that the acquisition of encoding sequences for these enzymes by horizontal transfer enhanced the ability of early plants to colonise the land. The improved ability to sequester and release inorganic phosphate for carbon fixation by photosynthetic algae in the ocean may have accelerated or even triggered global glaciation events. There is some evidence for DNA sequences encoding PPKs in a wider range of eukaryotes, notably some invertebrates, though it is unclear that these represent functional genes.Polyphosphate (poly P) is found in all cells, carrying out a wide range of essential roles. Studied mainly in prokaryotes, the enzymes responsible for synthesis of poly P in eukaryotes (polyphosphate kinases PPKs) are not well understood. The best characterised enzyme from bacteria known to catalyse the formation of high molecular weight polyphosphate from ATP is PPK1 which shows some structural similarity to phospholipase D. A second bacterial PPK (PPK2) resembles thymidylate kinase. Recent reports have suggested a widespread distribution of these bacteria type enzymes in eukaryotes.ResultsOn – line databases show evidence for the presence of genes encoding PPK1 in only a limited number of eukaryotes. These include the photosynthetic eukaryotes Ostreococcus tauri, O. lucimarinus, Porphyra yezoensis, Cyanidioschyzon merolae and the moss Physcomitrella patens, as well as the amoeboid symbiont Capsaspora owczarzaki and the non-photosynthetic eukaryotes Dictyostelium (3 species), Polysphondylium pallidum and Thecamonas trahens. A second bacterial PPK (PPK2) is found in just two eukaryotes (O. tauri and the sea anemone Nematostella vectensis). There is some evidence for PPK1 and PPK2 encoding sequences in other eukaryotes but some of these may be artefacts of bacterial contamination of gene libraries.ConclusionsEvidence for the possible origins of these eukaryote PPK1s and PPK2s and potential prokaryote donors via horizontal gene transfer is presented. The selective advantage of acquiring and maintaining a prokaryote PPK in a eukaryote is proposed to enhance stress tolerance in a changing environment related to the capture and metabolism of inorganic phosphate compounds. Bacterial PPKs may also have enhanced the abilities of marine phytoplankton to sequester phosphate, hence accelerating global carbon fixation.

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate.

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii 210A was purified 200-fold to apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate. No activity was observed with other polyphosphates and a wide variety of organic phosphate esters. The molecular mass of the enzyme was estimated to be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit composition of six identical polypeptides with a molecular mass of 23 kDa. The cation Mg2+ was required for activity, the activity with Mn2+, Co2+ and Zn2+ was 48, 48 and 182% of the activity observed with Mg2+, respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent Km for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.

Differences in polyphosphate accumulation and phosphate adsorption by Acinetobacter isolates from wastewater producing polyphosphate: AMP phosphotransferase

Ten isolates of Acinetobacter spp, obtained from sewage and showing high activities of polyphosphate: AMP phosphotransferase [ranging from 10 to 57 nmol min −1 (mg protein −1)] were studied for their differences in polyphosphate accumulation. Using electron microscopy, differences in numbers and sizes of the stored granules for the different strains were observed. These granules could be unambiguously identified as polyphosphate granules by electron spectroscopic imaging (ESI). Size and numbers of these granules found in different strains correlated with the intensity of different bands obtained from fractionated polyphosphates by polyacrylamide gel-electrophoresis (PAGE). Two different fractions, both consisting of high molecular weight polyphosphates (equivalent chain length), were obtained. Their distribution was strain specific. By electron microscopy and the ESI technique it could be further shown that all strains produced extracellular polymers (ECP). For one strain, a considerable phosphate adsorption at these ECP was observed. These data indicate that even in pure culture studies, phosphate adsorption must be considered in addition to the phosphate metabolism of specialized organisms for a sufficient explanation of the excess “biological” phosphate removal. For the much more complex situation in sewage treatment plants it is indispensable to consider various biological and chemical mechanisms for a sufficient explanation of “biological phosphate removal”.

Contribution of precipitated phosphates and acid-soluble polyphosphate to enhanced biological phosphate removal

The amount of precipitated metal phosphates and the acid-soluble polyphosphate concentration were measured in several sludges and pure cultures of Acinetobacter strains. The percentage of metal phosphates of the total phosphorus content of the sludges varied between negligible and about 80%. Increasing ability to release phosphate anaerobically was frequently parallelled by a decreasing contribution of precipitated metal phosphates to the total phosphorus concentration in 10 different sludges. Sludges from fill and draw systems with over 100 mg P/g dry wt, were almost completely devoid of these precipitates. The same was true for polyphosphate-accumulating cultures of Acinetobacter. Besides high molecular weight polyphosphate, the sludges also contained low polymeric polyphosphates (LPP), ranging from 0 to 50% of the total phosphate content. The LPP fraction in polyphosphate-accumulating cultures of Acinetobacter strains was between 3 and 23% of their total phosphate content. Sludges developed in the fill and draw systems could release more than 50% of the accumulated phosphate within 60 min. About 66% of this phosphate originated from LPP. Pure cultures of Acinetobacter strains released less than 1 mg P/g dry wt in the same time. Reduction of phosphate release by an increase of the pH in the medium as a result of denitrification could only partly be explained by formation of precipitated phosphates.

Observations relating to enhanced phosphorus removal in biological systems

In this work chemical fractionation and 31P-NMR spectroscopy were employed to explore certain unique characteristics of activated sludge exhibiting enhanced phosphorus removal. Two sequencing batch reactors were used in this research. The two reactors have been maintained in an environmental chamber and operated under the same conditions for well over 3 years. The only difference between the reactor systems is one system receives a portion of its organic carbon in the dissolved form as glucose (D-reactor) while the second reactor (P-reactor) receives an equivalent amount of organic material in the particulate form as starch. Chemical fractionation data and 31P-NMR analysis indicate that in the D-reactor, during periods of enhanced P-removal, transient phosphate is stored primarily as high molecular weight polyphosphates and nucleic acids. A portion of the transient polyphosphate is located outside the cytoplasmic membrane and is cell bound. No polyphosphate was found to exist in the mobile form in the cytoplasm. On the other hand, similar analysis on sludge taken from the P-reactor, during periods of enhanced P-removal, indicate that transient phosphate is stored primarily as low molecular weight polyphosphate. The transient polyphosphate is located inside the cytoplasmic membrane. A portion of the polyphosphate is cell bound while the remainder exists in the mobile form in the cytoplasm. Kinetic analysis suggests the possibility of a chemical intermediate(s) being formed in the degradation of polyphosphate to orthophosphate in sludge from the D-reactor while in sludge from the P-reactor the degradation process appears to proceed directly from polyphosphate to orthophosphate.

A 31P nuclear magnetic resonance study of Crithidia luciliae

31P NMR has been used to observe phosphorus-containing compounds in both perchloric acid and KOH extracts and in whole cell suspensions of Crithidia luciliae. Cells were grown in Bone and Steinert medium, or in a modified RPMI culture medium and harvested after about 72 h in mid- to late log phase. 31P NMR spectra of the perchloric acid extracts indicated that 3-phosphoglycerate, which is normally at low concentrations in most cells, was the dominant phosphorus-containing compound in the sugar phosphate region. 3-Phosphoglycerate is the end product of glycosomal glycolysis and our finding is consistent with previous observations of the failure to detect prior glycolytic intermediates. Other metabolites observed were ATP, ADP, NAD(P) +, phosphoenolpyruvate and low molecular weight polyphosphates (PP n, n<20). The presence of high molecular-weight polyphosphates was established by spectra recorded on extracts obtained through subsequent treatment of the insoluble fraction with KOH. 31P NMR experiments on whole cells indicated that the average main internal pH of cells in late-log growth phase was approx. pH 7.2±0.1, using the orthophosphate resonance as an indicator. The cells responded to the addition of glucose (final concentration approx. 35 mM) with a decrease in pH, both internal (ΔpH=−0.9 (55 min) −1) and external (ΔpH=−1.3 (15 min) −1). Polyphosphates and ATP could not be observed in whole cell experiments, although perchloric acid extracts of identically treated cells showed no significant depletion of these compounds.

Subcellular Phosphorus Kinetics for Lake Ontario Plankton

The subcellular distribution of recently incorporated 32PO4 was used to demonstrate that plankton in the surface waters of Lake Ontario were phosphorus deficient to some extent throughout the entire study period from April to October 1982. Most (~80%) of the initial uptake of carrier-free radioactive PO4 was in the low molecular weight (LMW) fraction. Ribonucleic acid (RNA) was the other major fraction. Although uptake was often complete within 1 h, changes in the subcellular distribution continued for 6–20 h with about half the LMW32P being transferred to RNA. Both a phospholipid fraction and a fraction extracted with hot trichloroacetic acid (HTCA) containing DNA and high molecular weight polyphosphate increased to 4–15% of the total isotope incorporated. In contrast with these experiments where net P uptake was zero, with added PO4 the net influx continued for periods in excess of 30 h but the subcellular distribution was constant within 2–4 h. About 80% of the initial uptake was as LMW32P and a rapid net synthesis of LMW polyphosphate was observed. These patterns were common for both small and large plankton size classes.

Polyphosphate kinase from Propionibacterium shermanii. Demonstration that the synthesis and utilization of polyphosphate is by a processive mechanism.

The mechanism of synthesis of inorganic polyphosphate by polyphosphate kinase (EC 2.7.4.1) from Propionibacterium shermanii is shown to be processive. Analysis of the synthesized polyphosphate on polyacrylamide gels, which resolve on the basis of molecular weight, proves that the elongation reaction occurs without dissociation of intermediate sizes of the polymer from the enzyme. As a consequence, only high molecular weight polyphosphates are synthesized. The mechanism is processive both in the presence and absence of basic protein. It has been shown previously that basic proteins stimulate the synthesis of polyphosphate (Robinson, N.A., Goss, N.H., and Wood, H.G. (1984) Biochem. Int. 8, 757-769). In addition, using a similar method, it is shown that the reverse reaction, the utilization of polyphosphate to phosphorylate ADP, occurs by a processive mechanism. Accordingly, polyphosphates formed by polyphosphate kinase in the cell would be entirely high molecular weight.

Isolation and characterization of polyphosphates from the yeast cell surface

When cells of Saccharomyces fragilis are subjected to osmotic shock, they release a limited amount of inorganic polyphosphate into the medium, which represents about 10% of the total cellular content. The osmotic shock procedure causes no substantial membrane damage, as judged from the unimpaired cell viability, limited K + leakage and low percentage of stained cells. It is therefore suggested that this polyphosphate fraction is localized outside the plasma membrane. The released polyphosphate fraction differs from the remaining cellular polyphosphates in two respects: the mean chain length of the shock-sensitive fraction is significantly higher than that of the total cellular polyphosphates and its metabolic turnover rate, subsequent to pulsing with [ 32P]orthophosphate is much lower compared to the rest of the cellular polyphosphate. Incubation of intact cells with the anion exchange resin Dowex AG 1-X4 results in the release of high molecular weight polyphosphates. These results suggest that the osmotic shock-sensitive polyphosphate fraction has specific characteristics in both its cellular localization and metabolism.

Isolation of a high molecular weight polyphosphate from Neisseria gonorrhoeae.

Neisseria gonorrhoeae, as well as other Neisseriae, produce polyphosphate. This polyphosphate exists in two forms. Approximately half of it is loosely associated with the cells and can be recovered by washing in neutral buffers under conditions in which no significant lysis of the cells is observed. The other half is either intracellular or tightly associated, because it requires digestion of the cells with perchloric acid or sodium hypochlorite. Polyphosphate obtained by both methods was purified by column chromatography and chemically characterized. In contrast to other organisms, gonococci do not respond with increased polyphosphate synthesis when shifted from phosphate starvation to a phosphate-rich medium. In addition, gonococcal polyphosphate does not serve as a depletable phosphate source during phosphate starvation. All strains of Neisseriae examined produce substantial amounts of polyphosphate.

Polyphosphate Metabolism in Micro-Organisms

Publisher Summary This chapter discusses the physiological role of inorganic polyphosphates. Inorganic polyphosphates are linear polymers in which orthophosphate residues are linked by energy-rich phospho-anhydride bonds. Inorganic polyphosphates have been found in almost all tested representatives of living cells. They have been detected in eubacteria, fungi, algae, mosses, protozoa, insects, and in various tissues of higher plants and animals. In highly organized organisms, polyphosphates perform several specialized functions, being donors of activated phosphate only for quite specific biochemical or physiological processes. The chapter presents new aspects of inorganic pyrophosphate's metabolic and physiological role. Pyrophosphate is only a byproduct of numerous reactions of pyrophosphorolysis involved in biosynthesis of proteins, nucleic acids, lipids, polysaccharides, and nucleoside coenzymes. The energy-dependent synthesis of pyrophosphate during photosynthetic and oxidative phosphorylation is also discussed. The first evidence for an energy-dependent biosynthesis of pyrophosphate is obtained with animal tissue homogenates. Finally, several modern concepts about the role of high molecular-weight polyphosphates and pyrophosphate in evolution of phosphorus metabolism are presented in the chapter.

Polyphosphate accumulation in sulphur-starved cells ofRhodella maculata

Rhodella cells grown without added sulphate (-S) divide at a slower rate and secrete sulphated polysaccharide into the medium at a slower rate than control cells. Light and electron microscopy of 20 day -S grown cells show the presence of large numbers of membrane-bound cytoplasmic inclusions with the staining characteristics of polyphosphates (PP). Chemical extraction reveals increases in low and high mol. wt PP in the -S grown cells. The high molecular weight PP has been extracted by the phenol detergent technique and separated from nucleic acids by polyacrylamide gel electrophoresis.

Synthesis of high molecular weight polyphosphate with a partially purified enzyme from Salmonella.

SUMMARY: Polyphosphate (polyP) kinase was prepared from EDTA-lysozyme spheroplasts of a Salmonella minnesota rough mutant. The enzyme, purified about 8o-fold, was free of ATPase and polyP-degrading activity and contained neither nucleic acids nor polyP which might act as primer. Using this enzyme, 32P-labelled polyP was prepared from [32 γP]ATP. The polyP was identified by the action of degrading enzymes, phenol extraction, adsorption characteristics on charcoal and hydrolysis kinetics. Sedimentation data in sucrose gradients (S = 11·5) and gel filtration on agarose indicated a molecular weight in the range 300,000 to 400,000 daltons.

Inorganic polyphosphates in the wheat stem rust fungus and in rust-infected wheat leaves

Results presented in this paper establish the presence of inorganic polyphosphates in saprophytic cultures of the wheat stem rust fungus ( Puccinia graminis tritici), and in non-infected and rust- infected primary leaves of wheat. Evidence for the presence of these compounds was based on acid-lability, non-adsorption to charcoal, metachromatic reaction with toluidine blue, paper chromatographic mobility and histochemical staining. Ten days after inoculation, rust-infected primary leaves of wheat contained eight times more inorganic polyphosphate than control leaves of the same age. Much of this polyphosphate was located in the stem rust uredospores. Further analysis of the uredospores revealed that approximately 90% of the cold acid-soluble phosphorus which was not adsorbed to charcoal was in the form of inorganic polyphosphate. The corresponding figure for the saprophytic rust mycelium was 80%. Rust-infected wheat leaves and saprophytic mycelium contained both high and low molecular weight polyphosphates. The low molecular weight polyphosphates were heterogeneous with respect to electrophoretic mobility on polyacrylamide gels. In contrast, the polyphosphates of non-infected wheat leaves were of low molecular weight only and were relatively homogeneous.

Synthesis of polyphosphate by rat liver mitochondria

The synthesis and hydrolysis of acid insoluble polyphosphates has been observed in many lower species and the enzymes involved have been partially purified by Kornberg and Hughes. Data also exists indicating that in bacteria under experimental conditions of biosynthetic blockade, e.g., nitrogen deficiency etc., polyphosphates accumulate within cells. Lehninger and others have noted that mammalian mitochondria swell in the presence of mitochondrial substrates and in the absence of ADP but shrink when ATP concentration is maintained at a high level. A reasonable explanation for these observations could be that excess substrate in the absence of adequate concentrations of nucleotide acceptor, activates the turnover of high energy phosphate into a “phosphagen” or polyphosphate pool at a very rapid rate, thus allowing ATP concentration to fall. The data in this report indicate that substrate does markedly stimulate phosphate uptake into a high molecular weight polyphosphate of rat liver mitochondria with a concomitant decrease in ATP concentration.

Studies on complex formation by means of ion selective membranes

Membrane electrodes have been used to investigate the activity suppressing action of polyphosphates towards Ca ++ ions in aqueous solution. The method, which is fully described, has allowed to determine the activity coefficients of sodium counterions of low and high molecular weight polyphosphates and their calcium complexing ability. The interaction of polyphosphate anions with their awn sodium counterions and with added Ca ++ ions has been interpreted as mainly due to strong Coulombic field acting in the neighbourhood of the highly charged anions.

THE ROLE OF WATER IN THE FORMATION OF SODIUM TRIPHOSPHATE BY CALCINATION

The formation of sodium triphosphate by calcination, below the sodium triphosphate fusion point, of various phosphate mixtures of over-all composition 5Na2O.3P2O5.xH2O was investigated. Water plays an important role, decreasing the amounts of pyrophosphate and high molecular weight polyphosphate impurities and increasing the reaction rate under certain conditions. It is suggested that water catalyzes the reactions by: (1) facilitating ionic diffusion, (2) hydrolyzing —P—O—P—linkages, and (3) assisting in the crystallization of sodium triphosphate. With ortho- and pyro-phosphate mixtures temperatures of about 300 °C. and higher are necessary for the rapid formation of sodium triphosphate. With glasses of the composition 5Na2O.3P2O5 good yields may be obtained at temperatures as low as 250 °C. when water is present.