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.     

Extraction and analysis of soluble inositol polyphosphates from yeast

Soluble inositol polyphosphates are implicated in the regulation of many important cellular functions. This protocol to extract and separate inositol polyphosphates from Saccharomyces cerevisiae is divided into three steps: labeling of yeast, extraction of soluble inositol polyphosphates and chromatographic separation. Yeast cells are incubated with tritiated inositol, which is taken up and metabolized into different phosphorylated forms. Soluble inositol polyphosphates are then acid-extracted and fractionated by high-performance liquid chromatography. The radioactivity of each fraction is determined by scintillation counting. This highly sensitive and reproducible method allows the accurate detection of subtle changes in the inositol polyphosphate profile and takes less than 48 h. It can easily be applied to other systems and we have included two adaptations of the protocol, one optimized for mammalian cells and the other for Arabidopsis thaliana.     

Identification of dinucleoside polyphosphates in adrenal glands

Dinucleoside polyphosphates have been characterised as extracellular mediators controlling numerous physiological functions like vascular tone or cell proliferation. Here we describe the isolation and identification of dinucleoside polyphosphates Ap(n)A (with n=2-3), Ap(n)G (with n=2-6) as well as Gp(n)G (with n=2-6) from adrenal glands. These dinucleoside polyphosphates are localised in granules of the adrenal glands. The dinucleoside polyphosphates diadenosine diphosphate (Ap(2)A), diadenosine triphosphate (Ap(3)A), adenosine guanosine polyphosphates (Ap(n)G) and diguanosine polyphosphates (Gp(n)G), both with phosphate group (p) numbers (n) ranging from 2 to 6, were identified by fractionating them to homogeneity by preparative size-exclusion- and affinity-chromatography as well as analytical anion-exchange and reversed-phase-chromatography from deproteinised adrenal glands and by analysis of the homogeneous dinucleoside polyphosphates containing fractions with post-source-decay (PSD) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). The identity of the dinucleoside polyphosphates was confirmed by retention time comparison with authentic dinucleoside polyphosphates. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5(')-positions of the riboses in all dinucleoside polyphosphates purified from adrenal glands. In conclusion, the identification of these dinucleoside polyphosphates in adrenal gland granules emphasises that these dinucleoside polyphosphates can be released from the adrenal glands upon stimulation into the circulation.     

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.     

Preparation of standards and determination of sizes of long-chain polyphosphates by gel electrophoresis

Procedures are presented for isolating fractions of long-chain polyphosphates which have a narrow range of sizes and for determining their chain lengths. The polyphosphates are isolated by elution from preparative polyacrylamide gels. Then, the lengths of these polymers are determined by a method of successive approximations of length from data obtained by electrophoresis on several different gels of varying polyacrylamide concentrations. Once sized, these isolated polyphosphates may be used as electrophoresis standards, making it possible to rapidly and accurately ascertain the size of other samples having unknown chain lengths. By comparison with two other procedures for sizing polyphosphates, it is shown that the method is definitely valid to a length of 450 and most likely to a length of at least 900. This electrophoresis procedure allows, for the first time, the determination of the range of sizes present and the average chain length with only 2-20 micrograms of polyphosphate.     

Diphosphoinositol polyphosphates: the final frontier for inositide research?

The diphosphoinositol polyphosphates comprise a group of highly phosphorylated compounds which have a rapid rate of metabolic turnover through tightly-regulated kinase/phosphohydrolase substrate cycles. The phosphohydrolases occur as multiple isoforms, the expression of which is apparently carefully controlled. Cellular levels of the diphosphoinositol polyphosphates are regulated by cAMP and cGMP in a protein kinase-independent manner. These inositides can also sense a specific mode of intracellular Ca2+ pool depletion. In this review, we will argue that these are characteristics of highly significant cellular molecules.     

On the nature of the granules of the genus spirillum

Summary Chemical analyses of centrifugal fractions of cell-free extracts of three spirilla and Sacch. cerevisiae showed that the granular inclusions accumulated by these organisms do not contain significant amounts (if any) of polyphosphates. Further, no trichloroacetic acid extractable-barium precipitable polyphosphates could be isolated from the spirilla. Thus, the granules of spirilla, heretofore called volutin, are not identifiable with polyphosphates.The phase opaque bodies present in members of this genus are composed primarily of poly--hydroxybutyrate. It is recommended that the term volutin no longer be used to describe granules showing a metachromatic reaction.     

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.     

Distribution of polyphosphates in cell-compartments of Chlorella fusca as measured by 31P-NMR-spectroscopy

In suspensions of the green alga Chlorella fusca the influence of high pH and high ethylene-diamine-tetraacetic acid concentrations in the external medium, of French-press and perchloric acid extraction of the cells and of alkalization of the intracellular pH on the polyphosphate signal in ³¹P-nuclear magnetic resonance (³¹P NMR) spectra was investigated.The results show that part of the polyphosphates of asynchronous Chlorella cells are located outside the cytoplasmic membrane and complexed with divalent metal-ions. These polyphosphates are tightly bound to the cell wall and/or the cytoplasmic membrane and are not susceptible to hydrolyzation by strong acid at room temperature, in contrast to the intracytoplasmic polyphosphates.Upon alkalization of the internal pH of Chlorella cells, polyphosphates, previously not visible in the spectra become detectable by ³¹P-NMR-spectroscopy. ³¹P-NMR spectroscopic monitoring of polyphosphates during gradual alkalization of the extra-and intracellular space is proposed as a quick method for the estimation of the cellular polyphosphate content and distribution.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - NTP/NDP Nucleotide triphosphate/-diphosphate - PCA Perchloric acid - ³¹P-NMR ³¹P-nuclear magnetic resonance - PolyP polyphosphates - PP₁, PP₂, PP₃ terminal, second and third phosphate residue of polyphosphates, respectively - PP₄ core phosphate residues of polyphosphates     

Effects of diadenosine polyphosphates on inward rectifier potassium currents in rat cardiomyocytes

Diadenosine polyphosphates are now considered a novel class of endogenous paracrine signal compounds. The putative role of these compounds in pathogenesis of myocardial infarction was proposed, since the concentration of diadenosine polyphosphates increases in the cardiac tissue following the ischemic lesion and myocardial necrosis. Therefore, possible effects of diadenosine polyphosphates on cardiac electrical activity and their ionic mechanisms are of considerable interest.     

Phosphorus-31 nuclear magnetic resonance study of polyphosphate metabolism in intact ectomycorrhizal fungi

Summary Phosphorus-31 nuclear magnetic resonance spectra at 36.4 MHz are presented for intact ectomycorrhizal fungi grown in pure culture. Resonances from polyphosphates and intracellular orthophosphate are identified inCenococcum graniforme, Hebeloma cylindrosporum, andH. crustuliniforme. Comparison of the NMR spectra with phosphorus fractionation of the fungi extracts leads to the statement that the NMR-observed polyphosphaes is a good part of the accumulated polyphosphates. In actively growing mycelia, this fraction account for up to 17% of total P.     

Polyphosphate glucokinase from Propionibacterium shermanii. Kinetics and demonstration that the mechanism involves both processive and nonprocessive type reactions

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate glucose phosphotransferase) has been partially purified (960-fold) from Propionibacterium shermanii. Throughout the purification, the ratio of polyphosphate glucokinase activity to ATP glucokinase activity remained approximately constant at 4 to 1. It is considered that both activities are catalyzed by the same protein. The mechanism of utilization of polyphosphate by polyphosphate glucokinase was investigated using polyphosphates of limited sizes that were isolated following gel electrophoresis of commercial heterogeneous polyphosphates. The results show that with long chain polyphosphates, the reaction proceeds by a processive type mechanism, and with short polyphosphates, it is nonprocessive. The Km for polyphosphate of chain length 724 is 2 X 10(-3) microM and increases with a decrease in chain length to 3.7 X 10(-2) microM at chain length 138. Subsequently, there is a very rapid increase of Km and at chain length 30 the Km is 4.3 microM. The rapid change in Km coincides with the shift in mechanism from the processive type mechanism in which there apparently is successive phosphorylation prior to release from the enzyme to a nonprocessive process in which the polyphosphate is released from the enzyme after each transfer. During the nonprocessive process, there is preferential utilization of the longer species. The Vmax is relatively constant with shorter polyphosphates but decreases with chain lengths longer than 347. In the cell, as a consequence of the low Km, the long chain polyphosphates probably are used preferentially to phosphorylate glucose.     

Human metastasis regulator protein H-prune is a short-chain exopolyphosphatase

The DHH superfamily human protein h-prune, a binding partner of the metastasis suppressor nm23-H1, is frequently overexpressed in metastatic cancers. From an evolutionary perspective, h-prune is very close to eukaryotic exopolyphosphatases. Here, we show for the first time that h-prune efficiently hydrolyzes short-chain polyphosphates (k cat of 3-40 s (-1)), including inorganic tripoly- and tetrapolyphosphates and nucleoside 5'-tetraphosphates. Long-chain inorganic polyphosphates (>or=25 phosphate residues) are converted more slowly, whereas pyrophosphate and nucleoside triphosphates are not hydrolyzed. The reaction requires a divalent metal cofactor, such as Mg (2+), Co (2+), or Mn (2+), which activates both the enzyme and substrate. Notably, the exopolyphosphatase activity of h-prune is suppressed by nm23-H1, long-chain polyphosphates and pyrophosphate, which may be potential physiological regulators. Nucleoside triphosphates, diadenosine hexaphosphate, cAMP, and dipyridamole (inhibitor of phosphodiesterase) do not affect this activity. Mutation of seven single residues corresponding to those found in the active site of yeast exopolyphosphatase led to a severe decrease in h-prune activity, whereas one variant enzyme exhibited enhanced activity. Our results collectively suggest that prune is the missing exopolyphosphatase in animals and support the hypothesis that the metastatic effects of h-prune are modulated by inorganic polyphosphates, which are increasingly recognized as critical regulators in cells.     

Effect of temperature on synthesis of polyphosphates in Yersinia pseudotuberculosis and Listeria monocytogenes under starvation conditions

It was found that at low temperature (6-8 degrees C) in the absence of nitrogen supply and at the presence of phosphate ions in the medium, Yersinia pseudotuberculosis and Listeria monocytogenes are able to actively synthesize reserve substances as polyphosphates. Most of the bacterial polyphosphates are alkali-soluble, especially at the preliminary stage of cell growth (lag-phase). This is proved by electron microscopic studies of ultrastructure of model microorganisms. During a long starvation period under conditions of carbon and energy source deficit, L. monocytogenes and Y. pseudotuberculosis consume this biopolymer for biosynthetic and bioenergetic processes.     

Localization of polyphosphates at the outside of the yeast cell plasma membrane

Under appropriate experimental conditions toluidine blue is bound to the yeast cell surface, without penetrating into the cells. Based on experimental observations it is highly probable that the dye is bound to polyphosphates, localized outside the plasma membrane. The probable localization of polyphosphates outside the plasma membrane is important in the context of the proposed involvement of polyphosphates in glucose transport in yeast.     

Inorganic polyphosphates of different fractions in the mutant yeast Saccharomyces cerevisiae with impaired mitochondrial ATP synthesis

Impaired synthetase function of the mitochondrial ATPase induced by mutation in the ATP22 gene results in decreased accumulation of inorganic polyphosphates in the stationary growth phase of the yeast Saccharomyces cerevisiae grown on glucose. The content of polyphosphates in the mutant strain in this phase is 2.5 times lower than in the parent strain. This difference is most pronounced for the acid-soluble polyP1 fraction and the alkali-soluble polyP3 fraction. Polyphosphate chain length in mutant cells is less than in the parent cells in both the acid-soluble polyP1 and in the salt-soluble polyP2 fractions. The mutation had no effect on polyphosphates content in the mitochondria.     

The metabolic properties of acid soluble polyphosphates in Saccharomyces cerevisiae

Summary Tripolyphosphate was found to be the predominant species of soluble polyphosphate in yeast. Evidence is presented which shows that under normal growth conditions tripolyphosphate had little or no turnover. The amounts of the various polyphosphates decreased as the chain length increased. Tetrapolyphosphate was shown to be synthesized more rapidly than tripolyphosphate. These observations suggest that short chain polyphosphates arise by degradation of longer chain length polyphosphates with tripolyphosphate the ultimate degradation product.During nitrogen starvation, the normal accumulation of tripolyphosphate rapidly ceased even though the cells continued normal growth for at least two hours. After the addition of L-amino acids or (NH₄)₂SO₄ to nitrogen starved cells, there was a dramatic increase in the accumulation of tripolyphosphate and tetrapolyphosphate which occurred at the same time as the increase in growth rate. Implications of this result are discussed in terms of possible functions of polyphosphate.     

The pho-controlled outer membrane porin PhoE does not contain specific binding sites for phosphate or polyphosphates

Purified PhoE-porins were reconstituted into black lipid bilayer membranes, and the selectivity and size of the reconstituted pores were determined. Addition of polyphosphates influenced the internal charge situation of the pore resulting in a shift from anion to cation selectivity. However, the pore size as judged from single channel conductances was not influenced by the addition of polyphosphates. A strong inhibition of the pore conductance only occurred when Mg2+ was also present in the aqueous phase. The inhibition of the pore function is presumably caused by the formation of a chelate between the divalent cation and the polyphosphate. Nevertheless, neither this inhibition nor the selectivity shift are specific to phosphate, because both effects can be mimicked by other polyvalent anions such as citrate. Inhibition of the PhoE pore function by polyphosphate in in vivo experiments confirmed the results of in vitro experiments that polyphosphate is only able to affect the permeability of the outer membrane toward beta-lactam antibiotics if Mg2+ is present. The outcome of the in vivo and the in vitro experiments are consistent with the assumption that the PhoE-porins do not contain a specific binding site for phosphate or polyphosphates but are anion selective because of an excess of positively charged amino acids inside or at the surface of the pore.     

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.     

Polyphosphate Inhibition of Growth of Pseudomonads From Poultry Meat

Both commercial polyphosphates and equivalent mixtures of chemically pure polyphosphates inhibited the growth of nonfluorescent pseudomonads in a synthetic medium. Fluorescent strains grew after a short lag. Inhibition was not caused by high pH, but rather by chelation of metal ions essential to the growth of the bacteria. Mg⁺⁺ and the natural competitive chelators, pyoverdine and bacteriological peptone, reversed the inhibition. Chilling chicken carcasses overnight in slush ice containing 3 and 8% polyphosphates lengthened subsequent shelf-life 17 and 25%, respectively. Chickens held in continuous contact with 3 and 8% solutions of polyphosphates during storage at 2.2 C kept 17 and 67% longer, respectively. Only fluorescent strains developed in the presence of 3 and 8% polyphosphates. Chickens held in antiseptic ice containing 8% polyphosphates kept 60% longer than did those in water ice.