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At least 19 recordsLinked to original sources

PubMed1987

Polyphosphate kinase from Propionibacterium shermanii. Demonstration that polyphosphates are primers and determination of the size of the synthesized polyphosphate.

Polyphosphate kinase from Propionibacterium shermanii was purified to 70% homogeneity and shown to be a monomeric enzyme of molecular weight 83,000 +/- 3,000. It was demonstrated that short chains of polyphosphate serve as primers by using [32P]polyphosphate, 6-80 residues in length for synthesis of long-chain polyphosphate glucokinase, the radiolabel was found to be at the end of the polymer, proving that the mechanism of elongation of polyphosphate by polyphosphate kinase is strictly processive. Only 1 out of 3-8 of the polyphosphate chains contained the primer, indicating that there is a second unknown pathway of initiation which does not involve the polyphosphate primer. The termination of polyphosphate synthesis was investigated. With polyphosphate as a primer, the majority of the synthesized polyphosphate was 750 residues in length. With phosphate, in place of the polyphosphate primer, the major portion was about 2,000 residues in length but there was a large span of chain lengths down to 300. Termination is influenced by pH, temperature, and the concentration of the polyphosphate primer, with the chain length decreasing as either the temperature or the concentration of primer is increased.

Adenosine Triphosphate↗

PubMed1999

Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates.

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.

Acid Anhydride Hydrolases↗

PubMed2004

Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography.

Polyphosphate is ubiquitous and has a variety of biochemical functions. Among polyphosphate quantification methods, an enzymatic assay using Escherichia coli polyphosphate kinase (PPK), in which polyphosphate is converted to adenosine 5'-triphosphate and quantified by luciferase assay, is the most specific and most sensitive. However, chain-length specificity of the assay has not been analyzed in detail so far. Ion chromatography equipped with an on-line hydroxide eluent generator enabled us to analyze polyphosphate up to 50 inorganic phosphate (P(i)) residues, and we employed this method to investigate the chain-length specificity of PPK in this study. Several fractions of short-chain polyphosphate were prepared by electrophoresis, and the chain-length distribution was analyzed before and after 1-6 h PPK reaction by ion chromatography. Polyphosphates longer than 23 P(i) residues were processed by PPK completely after 1 h incubation, but complete processing of those between 11 and 22 P(i) residues required 6h incubation. Limited processing of polyphosphates of 10 P(i) residues or shorter were observed even after 6h incubation. Metachromasy of Toluidine blue O, an alternative method for polyphosphate quantification, showed broader chain-length specificity although it was not as sensitive as the enzymatic assay. Combination of these two methods would be practically applicable to analysis of polyphosphate dynamics in living organisms.

Chromatography, Ion Exchange↗

PubMed2001

Polyphosphate:AMP phosphotransferase and polyphosphate:ADP phosphotransferase activities of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa PAO1, we have found massive polyphosphate:AMP phosphotransferase activity and polyphosphate:ADP phosphotransferase activity known as the reverse catalytic activity of polyphosphate kinase which participates in polyphosphate synthesis in the bacterium. Biochemical analysis using the partially purified polyphosphate:ADP phosphotransferase has revealed that it is independent of polyphosphate kinase and can function as polyphosphate-dependent nucleoside diphosphate kinase which most prefers GDP to the other three nucleoside diphosphates as a phospho-acceptor. It has been also demonstrated that polyphosphate:AMP phosphotransferase activity marked in the bacterium mainly originates from the combined action of the polyphosphate:ADP phosphotransferase described above and adenylate kinase. Both of the polyphosphate-utilizing activities require short polyP as a phospho-donor whose chain length is <75.

Base Sequence↗

PubMed2003

Synthetic, nondegradable diadenosine polyphosphates and diinosine polyphosphates: their effects on insulin-secreting cells and cultured vascular smooth muscle cells.

Diadenosine polyphosphates show a dissimilarity between their effects in static and perifusion experiments with respect to insulin release that may be due to degradation of the compounds. The aim was to investigate two nondegradable compounds of bisphosphorothioates containing a methylene or chloromethylene group (namely, diadenosine 5',5' "-(P(1),P(4)-dithio-P(2),P(3)-methylene)tetraphosphate and diadenosine 5',5' "-(P(1),P(4)-dithio-P(2),P(3)-chloromethylene)tetraphosphate), as mixtures of three or four diastereomers. Owing to their modified structures, these compounds are resistant to degradation (ectophosphodiesterases, diphosphohydrolases, and phosphorylases). Both compounds tested were minimally degraded (2%) even after 16 h when incubated with insulin-secreting (INS-1) cells. Additionally, diinosine polyphosphates (Ip(5)I and Ip(6)I), putative antagonists of diadenosine polyphosphates, were tested. By use of [(3)H]Ap(4)A, saturable binding sites for both diadenosine polyphosphate analogues were found in INS-1 cells, 3T3 preadipocyte cells, and vascular smooth muscle cells (VSMC) and for both Ip(5)I and Ip(6)I in INS-1 cells. The synthesized diadenosine polyphosphate analogues have the same affinity as Ap(4)A, whereas Ip(5)I and Ip(6)I inhibit binding at higher concentrations (10-100 microM). Insulin release was investigated in static experiments over 90 min in INS-1 cells. Insulin release was inhibited dose-dependently by both of the diadenosine polyphosphate analogues to the same degree as by Ap(4)A. The glucose-induced insulin release curve was not shifted to the right. Both compounds inhibit insulin release only at high (insulin stimulatory) glucose concentrations, e.g., 5.6 mM glucose. Ip(5)I and Ip(6)I antagonized Ap(5)A-mediated inhibition of insulin release. [(3)H]Thymidine incorporation into VSMC was not influenced by either synthetic diadenosine polyphosphate analogue, indicating that Ap(4)A does not act by itself in this case but (active) degradation products mediate the effect. The data indicate the following. (1) Since nondegradable compounds inhibit insulin release as well as Ap(4)A, it is Ap(4)A itself and not any of its degradation products that induces this effect. (2) Diadenosine polyphosphate effects on cell proliferation are mediated via a degradation product in contrast to their effect on insulin release. (3) Ip(5)I and Ip(6)I act like antagonists. Both synthetic analogues and diinosine polyphosphates are valuable tools for diabetes research.

3T3 Cells↗

PubMed1997

Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor.

A new family of dinucleotide derivatives, diinosine polyphosphates, has been synthesized through the use of the enzyme 5' adenylic acid deaminase from Aspergillus sp., starting from the corresponding diadenosine polyphosphates. Functional studies were performed on rat brain synaptic terminals in which a dinucleotide receptor has been described that is specific for adenine dinucleotides. The results demonstrated that diinosine polyphosphates did not behave as agonists on the diadenosine polyphosphate receptor (also know as P4 purinoceptor), but they were very efficient as antagonists in abolishing the Ca2+ responses elicited by diadenosine pentaphosphate. The IC50 values for diinosine triphosphate, diinosine tetraphosphate, and diinosine pentaphosphate were 4.90 +/- 0.10 microM, 8.33 +/- 0.22 microM, and 4.23 +/- 0.12 nM, respectively. The diinosine polyphosphates also antagonized the ATP receptors present in synaptic terminals, showing IC50 values of 100.08 +/- 5.72 microM for diinosine triphosphate, 29.51 +/- 1.40 microM for diinosine tetraphosphate and 27.75 +/- 1.65 microM for diinosine pentaphosphate. The antagonistic ability of these diinosine nucleotides was studied in comparison with other P1 and P2 purinoceptor antagonists, such as suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, and 8-cyclopentyl-1,3-dipropylxanthine. These purinergic antagonists did not inhibit the response of the P4 purinoceptor; only the diinosine polyphosphates were able to act as antagonists on the dinucleotide receptor. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid attenuated the responses elicited by ATP, as did the diinosine polyphosphate compounds. The most antagonistic diinosine polyphosphate for the dinucleotide and ATP receptors was diinosine pentaphosphate, which was 6000 times more selective for the P4 purinoceptor than it was for the ATP receptor.

Animals↗

PubMed1986

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.

Kinetics↗

PubMed1984

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.

Electrophoresis, Polyacrylamide Gel↗

PubMed1980

Detection of polyphosphates and enzymes of polyphosphate metabolism in Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus cells, parasitizing in E. coli, contain a considerable amount of inorganic polyphosphates, 55% of the total pool of which is due to the most polymeric acid-insoluble polyphosphates. B. bacteriovorus contains enzymes participating both in the synthesis and consumption of polyphosphates, i.e. 1,3-diphosphoglycerate: polyphosphate phosphotransferase, polyphosphate glucokinase, polyphosphatase, tripolyphosphatase, pyrophosphatase, acid and alkaline phosphatases. The possible role of high-molecular polyphosphates in the vital activity of the bacterial parasite B. bacteriovorus is discussed.

Bdellovibrio↗

PubMed1987

The mechanism of utilization of polyphosphate by polyphosphate glucokinase from Propionibacterium shermanii.

The phosphorylation of glucose by polyphosphate glucokinase with both long- and short-chain polyphosphates has been shown to occur by either a nonprocessive mechanism, i.e. with repeated association and dissociation of the polyphosphate from the enzyme after each phosphorylation or by a quasiprocessive mechanism in which several phosphorylations occur prior to the release of polyphosphate and the reassociation with the enzyme. In contrast, the phosphorylation of ADP to ATP by polyphosphate kinase is by a strictly processive mechanism; the phosphorylation occurs without release of the polymer from the enzyme prior to termination of the reaction (Robinson, N. A., Clark, J. E., and Wood, H. G. (1987) J. Biol. Chem. 262, 5216-5222). The demonstration that the mechanism is quasi-or nonprocessive was accomplished by electrophoresis using a variety of concentrations of polyacrylamide gels which made it possible to detect the intermediate sizes formed during the reactions. It also has been shown that all chains longer than about 100 are used simultaneously, but with chains of less than 100 residues, there is preferential utilization of the longest chains. Thus a narrow range of sizes is formed from a heterogeneous mixture of long chains. It is this formation of the narrow range of sizes that makes it possible to use polyphosphate glucokinase for the determination of the average size of long chains (Pepin, C. A., Wood, H. G., and Robinson, N. A. (1986) Biochem. Int. 12, 111-123).

Adenosine Diphosphate↗

PubMed1978

Interaction of phenosafranine with nucleic acids and model polyphosphates. I. Self-aggregation and complex formation with inorganic polyphosphates.

Aggregation of phenosafranine in concentrated aqueous solutions and interaction with polyphosphates was studied by absorption and fluorescence spectroscopy. At concentrations greater than 10(-3)M phenosafranine forms dimers (Kd = 3.8 x 10(2) 1.mole-1), which are characterized by a hypsochromic shift of the visible and near ultraviolet absorption maxima accompanied by a hypochromic effect. No fluorescence could be detected from phenosafranine dimers. Analogues spectra changes were observed when a polyphosphate was titrated with phenosafranine, which indicated that with increasing saturation of the polyphosphate binding sites phenosafranine gradually became bound in the aggregated form. Full saturation of the polyphosphate binding sites with phenosafranine was reached only when an excess of free dye was present. The cooperative binding of phenosafranine to a polyphosphate could be evaluated by means of a theory proposed by Schwarz et al. At the zero ionic strength and at 25 degrees C the binding was characterized by cooperative binding constant K = 6.2 x 10(5) 1.mole-1, number of binding sites per monomeric phosphate residue g = 0.4, and cooperativity parameter q-30. Spectroscopic properties of phenosafranine in the aggregated and polyphosphate-bound states were compared with those of ethidium bromide.

Binding Sites↗

PubMed1978

Interaction of phenosafranine with nucleic acids and model polyphosphates. I. Self-aggregation and complex formation with inorganic polyphosphates.

Aggregation or phenosafranine in concentrated aqueous solutions and its interaction with polyphosphates was Studied by absorption and fluorescence spectroscopy. At concentrations > 10(-3) M phenosafranine forms dimers (Kd = 3.8 x 10(2) l.mole(-1)), which are characterized by a hypsochromic shift of the visible and near ultraviolet absorption maxima accompanied by a hypochromic effect. No fluorescence could be detected from phenosafranine dimers. Analogous spectral changes were observed when a polyphosphate was titrated with phenusafranine, which indicated that with increasing saturation of the polyphosphate binding sites phenosafranine gradually became bound in the aggregated form. Full saturation of the polyphosphate binding sites with phenosafranine was reached only when an excess of free dye was present. The cooperative binding of phenosafranine to a polyphosphate could be evaluated by means of a theory proposed by Schwarz et al. At the zero ionic strength and at 25 degrees C the binding was characterized by cooperative binding constant K = 6.2 x 10(5) l.mole(-1), number of binding sites per monomeric phosphate residue g = 0.4, and cooperativity parameter q reverse similar 30. Spectroscopic properties of phenosafranine in the aggregated and poly phosphate-bound stotes were compared with those of ethidium bromide.

Journal Article↗

PubMed1992

Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes do not affect polyphosphate metabolism.

The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.

Genes, Fungal↗

PubMed1995

Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants.

The pathogenic Neisseria species accumulate polyphosphate to levels between 10 and 20% of their total phosphate content. However, the significance of this compound for the growth and pathogenicity of these species is not understood. A previous report (C.R. Tinsley, B.N. Manjula, and E.C. Gotschlich, Infect. Immun. 61:3703-3710, 1993) describes the purification of polyphosphate kinase, the enzyme responsible for synthesis of polyphosphate, from Neisseria meningitidis BNCV. By use of probes based on the amino acid sequence of the purified enzyme, the structural gene ppk has been cloned and sequenced. The coding sequence is 2,055 bp long and codes for a protein of 77.2 kDa. The open reading frame of the cloned gene was interrupted by the insertion of a kanamycin resistance cassette, and ppk mutants were obtained in both Neisseria gonorrhoeae and N. meningitidis by transformation with the recombinant plasmid. Amounts of polyphosphate in the ppk mutants were reduced to between 2 and 10% of wild-type levels. The mutants grew less vigorously than wild-type organisms in vitro and showed a striking increase in sensitivity to killing by human serum.

Amino Acid Sequence↗

PubMed1986

Determination of the size of polyphosphates with polyphosphate glucokinase.

A procedure for determining the size of inorganic polyphosphates of chain lengths up to about 750 is described. It involves reducing the size with polyphosphate glucokinase to a chain length that can be accurately determined by polyacrylamide gel electrophoresis. This measurement along with determination of the glucose-6-P formed and the total phosphate of the original polyphosphate permits calculation of the chain length. The accuracy of this method has been demonstrated by comparison with other reliable procedures. Thus far, it is the only method available for sizing long chain polyphosphates with nmol quantities.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

Inorganic polyphosphate and polyphosphate kinase: their novel biological functions and applications.

In this review, we discuss the following two subjects: 1) the physiological function of polyphosphate (poly(P)) as a regulatory factor for gene expression in Escherichia coli, and 2) novel functions of E. coli polyphosphate kinase (PPK) and their applications. With regard to the first subject, it has been shown that E. coli cells in which yeast exopolyphosphatase (poly(P)ase), PPX1, was overproduced reduced resistance to H2O2 and heat shock as did a mutant whose polyphosphate kinase gene is disrupted. Sensitivity to H2O2 and heat shock evinced by cells that overproduce PPX1 is attributed to depressed levels of rpoS expression. Since rpoS is a central element in a regulatory network that governs the expression of stationary-phase-induced genes, poly(P) affects the expression of many genes through controlling rpoS expression. Furthermore, poly(P) is also involved in expression of other stress-inducible genes that are not directly regulated by rpoS. The second subject includes the application of novel functions of PPK for nucleoside triphosphate (NTP) regeneration. Recently E. coli PPK has been found to catalyze the kination of not only ADP but also other nucleoside diphosphates using poly(P) as a phospho-donor, yielding NTPs. This nucleoside diphosphate kinase-like activity of PPK was confirmed to be available for NTP regeneration essential for enzymatic oligosaccharide synthesis using the sugar nucleotide cycling method. PPK has also been found to express a poly(P):AMP phosphotransferase activity by coupling with adenylate kinase (ADK) in E. coli. The ATP-regeneration system consisting of ADK, PPK, and poly(P) was shown to be promising for practical utilization of poly(P) as ATP substitute.

Adenosine Triphosphate↗

PubMed2000

Inorganic polyphosphate kinase and adenylate kinase participate in the polyphosphate:AMP phosphotransferase activity of Escherichia coli.

Polyphosphate kinase (PPK), responsible for the processive synthesis of inorganic polyphosphate (polyP) from ATP in Escherichia coli, can transfer in reverse the terminal phosphate residue of polyP to ADP to yield ATP. PolyP also serves as a donor in a polyP:AMP phosphotransferase (PAP) activity observed in extracts of Acinetobacter johnsonii and Myxococcus xanthus. We have found that overexpression of the gene encoding PPK results in a large enhancement of PAP activity in E. coli. The PAP activity requires both PPK and adenylate kinase in equimolar amounts. PPK and adenylate kinase form a complex in the presence of polyphosphate. We discuss a phosphotransfer mechanism that involves both enzymes and enables polyP to be a phospho-donor to AMP.

Adenylate Kinase↗

PubMed2001

A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase.

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.

Journal Article↗