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PubMed1981

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.

Biological Transport, Active↗
PubMedDate not supplied

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.

Archaea↗
PubMed2006

Batch production of polyhydroxyalkanoate by low-polyphosphate-content activated sludge at varying pH.

Polyhydroxyalkanoate (PHA), a biodegradable plastic, can be produced from excess activated sludge by utilizing intracellular glycogen and polyphosphate as energy sources under growth-limiting conditions. Activated sludge of 2%, 6%, and 8% polyphosphate with similar glycogen content of 33% was investigated for batch PHA production by varying the pH values from 6 to 8. Acetate applied at 1000 mg COD/L was almost exhausted within 80 min of anaerobic stage. The remaining glycogen in the sludge was higher at a lower pH because of less energy used for acetate uptake. Highest PHA content of 51% was obtained from sludge with an 8% polyphosphate content at pH 8. PHA production occurred rapidly within the first 20 min, with a productivity rate of 2.19 g PHA/L-h. The results in this study indicate that PHA production by using activated sludge is a promising alternative to a typical pure culture approach.

Bacteria, Aerobic↗
PubMed2006

The structure of the exopolyphosphatase (PPX) from Escherichia coli O157:H7 suggests a binding mode for long polyphosphate chains.

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains I and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the "closed" state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an "open" state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.

Acid Anhydride Hydrolases↗
PubMed2005

In vitro detection and characterisation of a polyphosphate synthesising activity in the yeast Candida humicola G-1.

An in vitro detectable polyphosphate-synthesising activity was characterised using two independent assay systems in extracts of the yeast Candida humicola G-1. Its properties were similar to those of a range of bacterial polyphosphate kinase enzymes. PCR amplification of C. humicola genomic DNA using universal primers for bacterial polyphosphate kinase genes yielded a product whose translated sequence showed up to 34% amino acid similarity to the bacterial enzyme.

Amino Acid Sequence↗
PubMed1982

The influence of polyphosphate ions on the precipitation of calcium oxalate.

We studied the growth of calcium oxalate monohydrate (COM) crystals in stirred suspensions by following the changes in ionic conductivity of supersaturated solutions at 25 +/- 0.02C. At a pH of 6.9-7.2, in the presence of polyphosphate ion, crystallization proceeded by an initial rapid step followed by a markedly reduced growth rate as compared with that in pure supersaturated solution. The initial surge was reduced as the polyphosphate concentration reached 1.6 X 10(-6) M, which can be interpreted as evidence of either surface nucleation or the incomplete adsorption of inhibitor at the active growth sites. In spontaneous precipitation experiments made in the presence of polyphosphate ions, the formation of calcium oxalate dihydrate and trihydrate is favored together with appreciable coprecipitation of additive ions.

Adsorption↗
PubMed2001

Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase.

Inositol polyphosphate 5-phosphatases are central to intracellular processes ranging from membrane trafficking to Ca(2+) signaling, and defects in this activity result in the human disease Lowe syndrome. The 1.8 resolution structure of the inositol polyphosphate 5-phosphatase domain of SPsynaptojanin bound to Ca(2+) and inositol (1,4)-bisphosphate reveals a fold and an active site His and Asp pair resembling those of several Mg(2+)-dependent nucleases. Additional loops mediate specific inositol polyphosphate contacts. The 4-phosphate of inositol (1,4)-bisphosphate is misoriented by 4.6 compared to the reactive geometry observed in the apurinic/apyrimidinic endonuclease 1, explaining the dephosphorylation site selectivity of the 5-phosphatases. Based on the structure, a series of mutants are described that exhibit altered substrate specificity providing general determinants for substrate recognition.

Amino Acid Sequence↗
PubMed1996

Kinetic mechanisms of polyphosphate glucokinase from Mycobacterium tuberculosis.

Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using inorganic polyphosphates [poly(P)] or ATP. The steady-state kinetic mechanisms of the poly(P)- and ATP-dependent glucokinase reactions were investigated using initial velocity, product inhibition, and dead-end inhibition analyses. In the poly(P)-dependent reaction, the enzyme follows an Ordered Bi Bi sequential mechanism with poly(P) binding to the enzyme first and glucose 6-phosphate dissociating last. Polyphosphate is utilized nonprocessively with a preference for longer chains due to higher kcat/K(m) values. The lack of inhibition at high poly(P) concentrations suggests that binding of poly(P) as a product is not favorable. In the ATP-dependent glucokinase reaction, the data are also consistent with an Ordered Bi Bi sequential mechanism, with ATP binding to the enzyme first and glucose 6-phosphate leaving last. At high concentrations, ATP displays competitive substrate inhibition with respect to glucose, which is consistent with the formation of an enzyme.ATP.ATP nonproductive complex. The overall catalytic efficiencies (kcat/KiaK(b)) of the poly(P)- and ATP-dependent reactions are approximately 10(11) M-2 s-1 and approximately 10(8) M-2 s-1, respectively. The higher catalytic efficiency, high value of the substrate specificity constant (kcat/K(a)) approaching a diffusion-controlled limit, and the absence of substrate inhibition in the poly(P)-dependent reaction suggest that poly(P), rather than ATP, is the major phosphate donor for poly(P)-glucokinase in M. tuberculosis.

Adenosine Triphosphate↗
PubMed1996

Mutation of the conserved domains of two inositol polyphosphate 5-phosphatases.

Two short amino acid motifs, WXGDXNXR and PXWCDRXL, define a large family of inositol polyphosphate 5-phosphatases. We tested the importance of seven of these conserved amino acids to substrate binding and catalysis by mutating each to alanine in the platelet 75 kDa inositol polyphosphate 5-phosphatase II (5-phosphatase II). Native and mutant forms of 5-phosphatase II were expressed in baculovirus-infected Sf9 cells, and the recombinant proteins were purified by Mono Q chromatography and studied for enzyme activity. Mutants D476A, N478A, D553A, and R554A had no detectable activity using all four known substrates for this enzyme. Mutants R480A, W551A, and I555A showed greatly reduced hydrolysis of Ins(1,4,5)P3 when compared to native enzyme [Km = 75 microM, Vm = 8300 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. Mutants W551A and I555A had a Km for Ins(1,4,5)P3 hydrolysis similar to that of the native enzyme (35 microM and 81 microM, respectively), suggesting that these amino acids do not play a role in binding substrate. By contrast, mutant R480A had both increased Km (634 microM) and decreased Vm [855 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. As judged by measurement of Km, mutant R480A retained normal binding of Ins(1,3,4,5)P4, suggesting that the arginine in motif 2 has a greater role in Ins(1,4,5)P3 binding than in Ins(1,3,4,5)P4 binding. Mutant I555A bound Ins(1,3,4,5)P4 with 8-fold reduced affinity. These mutations markedly reduced 5-phosphatase II hydrolysis of the three other substrates, Ins(1,3,4,5)P4, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. We also tested a mutation comparable to D553A, D460A, in the 110 kDa form of the signaling inositol polyphosphate 5-phosphatase (5SIP110). 5SIP110 D460A had no detectable enzyme activity but retained the ability to bind GRB2. These results are consistent with a role for these conserved amino acids in substrate binding and catalysis.

Amino Acid Sequence↗
PubMed2006

Synthesis and characterization of amphiphilic polyphosphates with hydrophilic graft chains and cholesteryl groups as nanocarriers.

Amphiphilic polyphosphate graft copolymers with varied densities of cholesteryl esters and hydrophilic graft chains were prepared, and the solution properties of the graft copolymers were evaluated. Polyphosphates were synthesized as backbones by ring-opening polymerization of 2-isopropyl-2-oxo-1,3,2-dioxaphospholane (IPP), 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl-2-bromoisobutyrate) (OPBB), and 2-choresteryl-2-oxo-1,3,2-dioxaphospholane (ChOP) using triisobutylaluminum as an initiator. Three types of polyphosphates (PIBr(x)Ch(y), x = number of OPBB units in a polymer; y = number of ChOP units in a polymer) such as PIBr4, PIBr6Ch1, and PIBr3Ch2 were obtained. The molecular weights of these polymers were 2.4 x 10(4), 2.4 x 10(4), and 2.6 x 10(4) g/mol, respectively. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was grafted from the OPBB sites in PIBr(x)Ch(y) via atom transfer radical polymerization (ATRP) in EtOH. In each polymer system, the molecular weight of the graft polymer was linear with conversion. Furthermore, the polymer radical concentration remained constant during polymerization; that is, the molecular weights of the graft chains were easily controllable with polymerization time. The solution properties of amphiphilic PIBr(x)Ch(y)-g-PMPCs were investigated by the methods of surface tension measurement, light scattering, and fluorescence probe. The transition point (cmc) of the surface tension of the PIBr(x)Ch(y)-g-PMPCs aqueous solution decreased with an increase in the number of ChOP units in a graft polymer. Particularly, PIBr3Ch2-g-PMPC14.9K formed nanosized associates (R(h) = 7.5 nm) with 2.2 molecules above 0.1 wt %. v79 cells were used to evaluate the cytotoxicity of the graft polymers, but no cytotoxicity was observed. The graft polymers containing cholesteryl groups effectively enhanced the solubility of paclitaxel in an aqueous solution.

Biopolymers↗
PubMedDate not supplied

Effect of polyphosphate metabolism on the Escherichia coli phosphate-starvation response.

A previously developed dynamic model of the Escherichia coli Pho regulon was extended to investigate the effect of polyphosphate synthesis and degradation on this control system. Differential equations for ATP and polyphosphate were formulated, and the model was applied to the growth of cells containing the ppk and ppx genes under control of separate, inducible promoters. In agreement with recent experimental observations, the degradation of polyphosphate by PPX during a period of phosphate limitation could repress the phosphate-starvation response. This is attributed to the release of phosphate from the cell into the periplasm, where it can be detected by the external phosphate sensor. A segregated model was then developed to account for differences in K(I), the dissociation constant for the repression complex, among cells of the population. Since K(I) is the key parameter in determining whether the Pho response is induced or repressed at a particular surface phosphate concentration, this permitted the induction of some cells while others remained repressed. The induction profiles resulting from the population-averaged values more closely matched experimental results than did those with the nonsegregated model.

Acid Anhydride Hydrolases↗
PubMed2000

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars.

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.

Animals↗
PubMed1997

Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa.

Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or over-abundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2-60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5' tetraphosphate.

Cell Membrane↗
PubMed1997

Signaling inositol polyphosphate-5-phosphatase. Characterization of activity and effect of GRB2 association.

An inositol polyphosphate-5-phosphatase (SIP-110) that binds the SH3 domains of the adaptor protein GRB2 was produced in Sf9 cells and characterized. SIP-110 binds to GRB2 in vitro with a stoichiometry of 1 mol of GRB2/0.7 mol of SIP-110. GRB2 binding does not affect enzyme activity implying that GRB2 serves mainly to localize SIP-110 within cells. SIP-110 hydrolyses inositol (Ins)(1,3,4,5)P4 to Ins(1, 3,4)P3. The enzyme does not hydrolyze Ins(1,4,5)P3 that is a substrate for previously described 5-phosphatases nor does it hydrolyze phosphatidylinositol (PtdIns)(4,5)P2. SIP-110 also hydrolyzed PtdIns(3,4,5)P3 to PtdIns(3,4)P2 as did recombinant forms of two other 5-phosphatases designated as inositol polyphosphate-5- phosphatase II, and OCRL (the protein that is mutated in oculocerebrorenal syndrome). The inositol polyphosphate-5-phosphatase enzyme family now is represented by at least 9 distinct genes and includes enzymes that fall into 4 subfamilies based on their activities toward various 5-phosphatase substrates.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Phosphoinositide-specific inositol polyphosphate 5-phosphatase IV inhibits Akt/protein kinase B phosphorylation and leads to apoptotic cell death.

Phosphoinositide-specific inositol polyphosphate 5- phosphatase IV has the affinity for PI(3,4,5)P(3) (K(m) = 0.65 microM) that is approximately 10-fold greater than the other inositol polyphosphate 5-phosphatases, which use this substrate including SHIP, OCRL, and 5ptase II, suggesting that it may be important in controlling intracellular levels of this metabolite. We created cell lines stably expressing the enzyme to study its effect on cell function. We found that overexpression of 5ptase IV in 293 cells caused the rapid depletion of both PI(4,5)P(2) and PI(3,4,5)P(3) in cells with corresponding increases in the products, PI(4)P and PI(3,4)P(2), changing the balance of two phosphoinositol products of phosphoinositide 3-kinase, PI(3,4)P(2) and PI(3,4,5)P(3), in the cell. One of the targets of these phosphoinositides is the serine/threonine kinase Akt, which plays an important role in the control of apoptosis. We were able to address the relative roles of PI(3,4)P(2) and PI(3,4,5)P(3) in the activation of Akt by selective depletion of these phosphoinositides in cells stably transfected with 5ptase IV and inositol polyphosphate 4-phosphatase (4ptase I). In cells transfected with 4ptase I, the level of PI(3,4)P(2) was reduced, and PI(3,4,5)P(3) was increased. Expression of the two enzymes had the opposite effect on the phosphorylation of Akt in response to stimulation with growth factors or heat shock. Akt phosphorylation was inhibited in cells expressing 5ptase IV but increased in 4ptase I cells and correlated with the intracellular level of PI(3,4,5)P(3) and not that of PI(3,4)P(2). The inhibition of Akt phosphorylation in cells expressing 5ptase IV makes them highly susceptible to FAS-induced apoptosis, whereas overexpressing of the 4ptase I protects cells from apoptosis. Our results place 5ptase IV as a relevant biological regulator of PI3K/Akt pathway in cells.

Apoptosis↗
PubMed2005

Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.

Adenosine Triphosphate↗
PubMed2000

Accumulation of polyphosphate granules in Helicobacter pylori cells under anaerobic conditions.

Helicobacter pylori is known to transform to coccoid forms which might be involved in faecal-oral transmission. When the bacteria enter the intestine, they encounter anaerobiosis that is unfavourable for growth. The effect of anaerobiosis was investigated to determine whether H. pylori is viable under such conditions. H. pylori in the late logarithmic growth phase transformed from spiral to coccoid forms when transferred to and incubated anaerobically in fresh medium. Acridine orange staining indicated that the viability of coccoid forms was significantly reduced, but still measurable even at day 5 or 7 of anaerobic culture. The cells retained low but significant levels of the major sigma factor RpoD at day 5 or 7 of anaerobic culture. The cellular structures of coccoid forms contained polyphosphate granules at day 1 and even at day 7 when incubated anaerobically, whereas only a few granules were observed under micro-aerobic conditions. Poor formation of polyphosphate granules in micro-aerobic cultures correlated particularly well with lower levels of acridine orange staining. These results suggest that acridine orange-positive anaerobic coccoid forms are viable to a certain extent and that polyphosphate may support this viability.

Acridine Orange↗
PubMed1991

The location of the polyphosphate-binding sites on cytochrome c measured by NMR paramagnetic difference spectroscopy.

Analyses of unimolecular electron self-exchange reactions provide a comparatively simple and direct approach to understanding biological electron transfer. Such studies are currently limited by a lack of well characterised aggregating systems. In the presence of sodium hexametaphosphate, cytochrome c forms stable protein aggregates as a result of binding hexametaphosphate at a single site on its surface (preceding paper in this issue of the journal). Here we report the location of the principal polyphosphate binding site on the surface of cytochrome c for both hexametaphosphate and a second polyphosphate, tripolyphosphate determined using 1H-NMR spectroscopy in conjunction with the relaxation probe potassium hexacyanochromium(III). Addition of either hexametaphosphate or tripolyphosphate to ferricytochrome c in the presence of the relaxation probe causes a decrease in intensity of several resonances in the paramagnetic difference spectrum, including Phe82 ortho/meta, Ile85 delta methyl and Ile9 gamma methyl. Together these effects put the site of polyphosphate binding close to lysines 13, 86, and 87. Additionally the effect of sodium tripolyphosphate and sodium trimetaphosphate on cytochrome c aggregation is described. The potential role of this site in anion-induced cytochrome c aggregation is discussed.

Animals↗