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PubMed1997

Molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate hydrolases and phosphorylases.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap4A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap4A hydrolase (Km = 0.8 microM) being sensitive to inhibition by fluoride (Ki = 24 microM) and adenosine 5'-tetraphosphate (Ki = 10 nM) and yielding ATP and AMP as products. A low Km Ap4A hydrolase (Km = 0.3 microM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of Km for Ap4A and Ki for adenosine 5'-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequences comparisons among eukaryotic diadenosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.

Acid Anhydride Hydrolases↗
PubMed2003

Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens.

To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.

Amino Acid Sequence↗
PubMed2003

Paralogous murine Nudt10 and Nudt11 genes have differential expression patterns but encode identical proteins that are physiologically competent diphosphoinositol polyphosphate phosphohydrolases.

We previously described paralogous human genes [NUDT10 and NUDT11 [where NUDT is (nucleoside diphosphate attached moiety 'X')-type motif, also known as the 'nudix'-type motif]] encoding type 3 diphosphoinositol polyphosphate phosphohydrolases (DIPP3) [Hidaka, Caffrey, Hua, Zhang, Falck, Nickel, Carrel, Barnes and Shears (2002) J. Biol. Chem. 277, 32730-32738]. Normally, gene duplication is redundant, and lacks biological significance. Is this true for the DIPP3 genes? We address this question by characterizing highly-conserved murine Nudt10 and Nudt11 homologues of the human genes. Thus these genes must have been duplicated prior to the divergence of primates and sciurognath rodents, approx. 115 million years ago, greatly exceeding the 4 million year half-life for inactivation of redundant paralogues; our data therefore indicate that the DIPP3 duplication is unusual in being physiologically significant. One possible functional consequence is gene neofunctionalization, but we exclude that, since Nudt10 and Nudt11 encode identical proteins. Another possibility is gene subfunctionalization, which we studied by conducting the first quantitative expression analysis of these genes. We demonstrated high Nudt10 expression in liver, kidney and testis; Nudt11 expression is primarily restricted to the brain. This differential, but complementary, expression pattern indicates that subfunctionalization is the evolutionary consequence of DIPP3 gene duplication. Our kinetic data argue that diphosphoinositol polyphosphates are more physiologically relevant substrates for DIPP3 than are either diadenosine hexaphosphate or 5-phosphoribosyl 1-pyrophosphate. Thus the significance of the Nudt10/Nudt11 duplication is specific hydrolysis of diphosphoinositol polyphosphates in a tissue-dependent manner.

Amino Acid Sequence↗
PubMed1987

Subsecond and second changes in inositol polyphosphates in GH4C1 cells induced by thyrotropin-releasing hormone.

It has been demonstrated previously that thyrotropin-releasing hormone (TRH) induces changes in inositol polyphosphates in the GH3 and GH4C1 strains of rat pituitary cells within 2.5-5.0 s. TRH also causes a rapid rise in cytosolic free calcium concentration ([Ca2+]i) in these cells which is due largely to redistribution of cellular calcium stores. Therefore, it has been concluded that TRH acts to release sequestered calcium in these cells via enhanced generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. If this conclusion were correct, TRH-enhanced accumulation of Ins(1,4,5)P3 should occur at least as rapidly as the increase in [Ca2+]i. We have shown previously that the rise in [Ca2+]i induced by TRH occurs within about 400 ms; thus, it was important to investigate the subsecond time-course of changes in inositol phosphates caused by TRH. Using a rapid mixing device, we have measured changes in inositol polyphosphates on a subsecond time scale in GH4C1 cells prelabelled with myo-[2-3H]inositol. Although TRH did alter inositol polyphosphate metabolism within 500 ms, the changes observed did not reveal a statistically significant increase in Ins(1,4,5)P3 within time intervals of less than 1000 ms. Thus, we have been unable to demonstrate that a TRH-induced rise in Ins(1,4,5)P3 precedes or occurs concomitantly with the rise in [Ca2+]i in GH4C1 cells. Although these results do not disprove the current view that Ins(1,4,5)P3 mediates the action of TRH on intracellular calcium redistribution, we conclude that caution should be exercised in this, and possibly other cell systems, in accepting the dogma that all of the rapid, agonist-induced redistributions of intracellular calcium are mediated by Ins(1,4,5)P3.

Animals↗
PubMed1996

Neutrophil apoptosis is delayed by the diadenosine polyphosphates, Ap5A and Ap6A: synergism with granulocyte-macrophage colony-stimulating factor.

In addition to ATP, platelets and other cell types can secrete high quantities of diadenosine polyphosphates Ap3A, Ap4A, Ap5A and Ap6A. There is increasing evidence to show that these molecules can function as novel modulators of cell function. For this report we have measured the effects of the diadenosine polyphosphates Ap5A and Ap6A on neutrophil apoptosis. These molecules can themselves delay neutrophil apoptosis (as assessed by morphology, function. CD16 expression and chromatin integrity), and are as effective on a molar basis as ATP, Ap3A and Ap4A. Moreover, these dinucleotides act synergistically with granulocyte-macrophage colony-stimulating factor (GM-CSF) to delay neutrophil apoptosis. Thus, diadenosine polyphosphates may act, in concert with cytokines, as novel modulators of neutrophil function and survival in certain types of inflammatory conditions.

Apoptosis↗
PubMed2002

The polyphosphate kinase plays a negative role in the control of antibiotic production in Streptomyces lividans.

The polyphosphate kinase gene (ppk) from Streptomyces lividans, which encodes a 774-amino-acid protein (86.4 kDa) showing extensive homology to other bacterial polyphosphate kinases, was cloned by polymerase chain reaction (PCR) using oligonucleotides derived from the putative ppk gene from the closely related species, Streptomyces coelicolor. In vitro, the purified Ppk was shown to be able to synthesize the polyphosphate [poly(P)] from ATP (forward reaction) as well as to regenerate ATP from the poly(P) in the presence of an excess of ADP (reverse reaction). In conditions of poly(P) synthesis, a phosphoenzyme intermediate was detected, indicating an autophosphorylation of the enzyme in the presence of ATP. The ppk gene was shown to be transcribed as a monocistronic mRNA from a unique promoter. Its transcription was only detectable during the late stages of growth in liquid minimal medium. A mutant strain interrupted for ppk was characterized by increased production of the antibiotic actinorhodin on rich R2YE solid medium (0.37 mM KH2PO4 added). This production was enhanced on the same medium with no KH2PO4 added but was completely abolished by the addition of 1.48 mM KH2PO4. In the ppk mutant strain, this increased production correlated with enhanced transcription of actII-ORF4 encoding the specific activator of the actinorhodin pathway. In that strain, the transcription of redD and cdaR, encoding the specific activators of the undecylprodigiosin and calcium-dependent antibiotic biosynthetic pathways, respectively, was also increased but to a lesser extent. The enhanced expression of these regulators did not seem to be related to increased relA-dependent ppGpp synthesis, as no obvious increase in relA expression was observed in the ppk mutant strain. These results suggested that the negative regulatory effect exerted by Ppk on antibiotic biosynthesis was most probably caused by the repression exerted by the endogenous Pi, resulting from the hydrolysis of the poly(P) synthesized by Ppk, on the expression of the specific activators of the antibiotic biosynthetic pathways.

Anti-Bacterial Agents↗
PubMed1995

Functional diversity of C2 domains of synaptotagmin family. Mutational analysis of inositol high polyphosphate binding domain.

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.

Amino Acid Sequence↗
PubMed1996

Inhibition of adenylyl cyclase by a family of newly synthesized adenine nucleoside 3'-polyphosphates.

The synthesis of a number of adenine nucleoside 3'-polyphosphates has been devised via a phosphotriester approach that combines the method of alkoxide activation with the use of 2,2,2-tribromoethyl phosphoromorpholinochloridate as a phosphorylating agent. The family of compounds included 3'ADP, 3'ATP, 2'-deoxy-3'ADP, 2'-deoxy-3'ATP, 2',5'-dideoxy-3'ADP, and 2',5'-dideoxy-3'ATP. Potency as inhibitors of adenylyl cyclases followed the order: 3'-mono- < 3'-di- < 3'-triphosphate and adenosine (Ado) < 2'-d-Ado < 2',5'-dd-Ado derivatives, with 2',5'-dideoxy-3'ATP exhibiting an IC50 of approximately 40 nM. This order was maintained with purified and recombinant forms of the type I enzyme. The nucleoside 3'-phosphates caused noncompetitive inhibition of the type I adenylyl cyclase from bovine brain, consistent with inhibition via the P-site. Inhibition was not due to hydrolytic products because this was minimal and inhibition kinetics by inorganic polyphosphates were inconsistent with those caused by the nucleoside 3'-polyphosphates. Only 3'ATP underwent cation-catalyzed, nonenzymatic hydrolysis, with the primary product being 2':3'-cAMP. Because 3'-ADP and 3'-ATP are naturally occurring, this class of compounds may physiologically regulate adenylyl cyclases and possibly other enzymes, mediating responses that include a reduction in 3':5'-cAMP levels and consequent reductions in protein kinase A-activated pathways.

Adenine Nucleotides↗
PubMed1999

Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85alpha Src homology 2 domains with phosphoinositides and inositol polyphosphates.

Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85alpha subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85alpha SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85alpha SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85alpha. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85alpha SH2 domains.

3T3 Cells↗
PubMed1999

The diadenosine hexaphosphate hydrolases from Schizosaccharomyces pombe and Saccharomyces cerevisiae are homologues of the human diphosphoinositol polyphosphate phosphohydrolase. Overlapping substrate specificities in a MutT-type protein.

Aps1 from Schizosaccharomyces pombe (Ingram, S. W., Stratemann, S. A. , and Barnes, L. D. (1999) Biochemistry 38, 3649-3655) and YOR163w from Saccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604-8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap(6)A and Ap(5)A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap(6)A and Ap(5)A, in preference to other diadenosine polyphosphates. The emergence of Ap(6)A and Ap(5)A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.

Acid Anhydride Hydrolases↗
PubMed2000

Discovery of molecular and catalytic diversity among human diphosphoinositol-polyphosphate phosphohydrolases. An expanding Nudt family.

The turnover of the "high energy" diphosphoinositol polyphosphates by Ca(2+)- and cyclic nucleotide-modulated enzymes is considered a regulatory, molecular switching activity. Target processes may include intracellular trafficking. Following our earlier identification of a prototype human diphosphoinositol-polyphosphate phosphohydrolase (hDIPP1), we now describe new 21-kDa human isoforms, hDIPP2alpha and hDIPP2beta, distinguished from each other solely by hDIPP2beta possessing one additional amino acid (Gln(86)). Candidate DIPP2alpha and DIPP2beta homologues in rat and mouse were also identified. The rank order for catalytic activity is hDIPP1 > hDIPP2alpha > hDIPP2beta. Differential expression of hDIPP isoforms may provide flexibility in response times of the molecular switches. The 76% identity between hDIPP1 and the hDIPP2s includes conservation of an emerging signature sequence, namely, a Nudt (MutT) motif with a GX(2)GX(6)G carboxy extension. Northern and Western analyses indicate expression of hDIPP2s is broad but atypically controlled; these proteins are translated from multiple mRNAs that differ in the length of the 3'-untranslated region because of utilization of an array of alternative (canonical and noncanonical) polyadenylation signals. Thus, cells can recruit sophisticated molecular processes to regulate diphosphoinositol polyphosphate turnover.

Acid Anhydride Hydrolases↗
PubMed2001

The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes.

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.

Acids↗
PubMed1991

Supplementation of broiler drinking water with liquid ammonium polyphosphate.

1. In each of two experiments lasting 21 days a commercially available ammonium polyphosphate solution was provided in the drinking water as the sole source of supplemental phosphorus for day-old chicks housed in batteries. 2. In both experiments, similar daily phosphorus intakes either from dicalcium phosphate in the food or from ammonium polyphosphate in the drinking water supported equivalent responses in body weight gains. The same was true for the tibia ash results in experiment 2. 3. Liquid ammonium polyphosphate, while marketed for addition to diets, appears to be also very effective as a waterborne source of phosphorus for broilers.

Animals↗
PubMed2001

Coronary vasomotor and cardiac electrophysiologic effects of diadenosine polyphosphates and nonhydrolyzable analogs in the guinea pig.

Platelet activation in heart disease is important owing to the effects of platelet-derived compounds on myocardial perfusion and cardiac electrophysiology. Diadenosine polyphosphates are secreted from platelets and present in the myocardium, but their electrophysiologic and vasomotor effects are incompletely understood. We used isolated guinea-pig hearts to study the effects of diadenosine triphosphate (Ap3A), tetraphosphate (Ap4A), pentaphosphate (Ap5A), and hexaphosphate (Ap6A) (10 pM-0.1 mM), comparing their actions to those of adenosine, adenosine triphosphate, and non-hydrolyzable Ap4A and Ap5A analogs. Diadenosine polyphosphates (0.1 nM-0.1 microM) transiently reduced coronary perfusion pressure, which recovered during the continued presence of the compounds. At concentrations greater than 0.1 microM effects were maximal and sustained (perfusion pressure decreased from 36.5+/-3.4 to 18.6+/-2.5 mm Hg, p < 0.001, with 1 microM Ap4A). The changes in action potential duration and refractory period developed slowly but were maintained (0.1 nM-1 microM). With 1 nM Ap4A, action potential duration increased from 170.6+/-2.6 to 187.3+/-3.8 ms, p < 0.05, and refractory period increased from 138.5+/-1.6 to 147.9+/-2.0 ms, p < 0.05. Ap4A and its analog reduced QRS duration (from 24.7+/-1.1 to 13.9+/-1.6 ms with 1 microM Ap4A, p < 0.05). P2-purinergic (adenosine triphosphate) receptor antagonism (suramin) reduced perfusion pressure but was without electrophysiologic effect. Other changes in coronary perfusion pressure and electrophysiologic variables associated with Ap4A were not seen in the presence of suramin. P1-(adenosine) antagonism (8-[p-sulfophenyl]theophylline) attenuated the electrophysiologic effects only. Diadenosine polyphosphates have potent cardiac electrophysiologic and coronary vasomotor effects via purinergic receptors, suggesting an important role during platelet activation in acute coronary syndromes.

Action Potentials↗
PubMed2004

Tissue engineered nucleus pulposus tissue formed on a porous calcium polyphosphate substrate.

STUDY DESIGN: This study describes the formation of nucleus pulposus tissue using a novel tissue engineering approach. OBJECTIVES: To determine if a construct composed of nucleus pulposus tissue on the surface of a calcium polyphosphate substrate could be formed in vitro with properties similar to native nucleus pulposus tissue. SUMMARY OF BACKGROUND DATA: There is no optimal treatment for the persistent pain associated with intervertebral disc degeneration. Disc replacement using artificial intervertebral discs has met with some success, and biologic transplantation is limited by the availability of donor tissues. METHODS: Nucleus pulposus cells were isolated from bovine caudal intervertebral discs. Cells were seeded at high density on the upper surface of a porous bone substitute material (calcium polyphosphate) and maintained up to 6 weeks in culture. In vitro formed tissue was compared to native nucleus pulposus for histologic appearance, biochemical composition (tissue cellularity, proteoglycan and collagen accumulation), and compressive mechanical properties. RESULTS: When maintained on the surface of a three-dimensional substrate, nucleus pulposus cells formed a continuous layer of tissue with a proteoglycan content equivalent to the native tissue. Although collagen accumulation attained only 26% than that of the native tissue, there was no difference in tissue stiffness, viscosity, or weight-bearing capacity of the in vitro formed tissue when compared with the native tissue. CONCLUSION: Nucleus pulposus-like tissue formed in vitro on the surface of a calcium polyphosphate substrate resembles the native tissue in terms of proteoglycan content and compressive mechanical properties. These studies are the first step toward developing a functional spinal unit in vitro.

Animals↗
PubMed1975

Purification and characterization of a polyphosphate kinase from Arthrobacter atrocyaneus.

Polyphosphate kinase, an enzyme which incorporated the gamma-phosphate of ATP into long-chain polyphosphate molecules, was purified more than 700-fold from Arthrobacter atrocyaneus by ammonium sulphate fractionation, DEAE-cellulose column chromatography and Ssphadex G-200 gel filtration. The enzyme had a broad pH optimum at 6-0 to 7-0 and required Mn2+ or Mg2+, histone, and inorganic phosphate for activity. The Km for Mn-ATP was 0-53 mM, and for inorganic phosphate was 1-67 mM. Free ATP concentrations greater than 8 muM inhibited the enzyme. Free Mn2+ or Mg2+ concentrations greater than 2 mM or 6 mM, respectively, were also inhibitory. Activity was strongly inhibited by 4 mM-ADP, 1 mM-PP1 or 20 mM-NaF. The effect of ADP might have resulted from reversing the equilibrium of the kinase reaction. The activation by phosphate ions might indicate a role for the enzyme in regulating intracellular phosphate levels or maintaining a phosphorus reserve. The level of enzymic activity in the bacteria responded to changes in inorganic phosphate concentration in the medium. Basic proteins, such as protamine, could substitute for histone as activator. Proteins such as casein or bovine serum albunim would also substitute for histone but only in the absence of inorganic phosphate. The presence of a protein might be necessary to form a complex with the product, thus preventing reversal of the reaction in vitro. The reaction product was characterized, and found to be labile in hydroxylamine, base, and acid at 100 degrees C. It behaved as a long-chain-polyphosphate molecule on chromatography in an Ebel's solvent. The enzymic activity was therefore not that of a protein kinase.

Adenosine Triphosphate↗
PubMed2004

A role of Arabidopsis inositol polyphosphate kinase, AtIPK2alpha, in pollen germination and root growth.

Inositol polyphosphates, such as inositol trisphosphate, are pivotal intracellular signaling molecules in eukaryotic cells. In higher plants the mechanism for the regulation of the type and the level of these signaling molecules is poorly understood. In this study we investigate the physiological function of an Arabidopsis (Arabidopsis thaliana) gene encoding inositol polyphosphate kinase (AtIPK2alpha), which phosphorylates inositol 1,4,5-trisphosphate successively at the D-6 and D-3 positions, and inositol 1,3,4,5-tetrakisphosphate at D-6, resulting in the generation of inositol 1,3,4,5,6-pentakisphosphate. Semiquantitative reverse transcription-PCR and promoter-beta-glucuronidase reporter gene analyses showed that AtIPK2alpha is expressed in various tissues, including roots and root hairs, stem, leaf, pollen grains, pollen tubes, the flower stigma, and siliques. Transgenic Arabidopsis plants expressing the AtIPK2alpha antisense gene under its own promoter were generated. Analysis of several independent transformants exhibiting strong reduction in AtIPK2alpha transcript levels showed that both pollen germination and pollen tube growth were enhanced in the antisense lines compared to wild-type plants, especially in the presence of nonoptimal low Ca(2+) concentrations in the culture medium. Furthermore, root growth and root hair development were also stimulated in the antisense lines, in the presence of elevated external Ca(2+) concentration or upon the addition of EGTA. In addition, seed germination and early seedling growth was stimulated in the antisense lines. These observations suggest a general and important role of AtIPK2alpha, and hence inositol polyphosphate metabolism, in the regulation of plant growth most likely through the regulation of calcium signaling, consistent with the well-known function of inositol trisphosphate in the mobilization of intracellular calcium stores.

Arabidopsis↗
PubMed1991

Hydrolysis of polyphosphates and permeability changes in response to osmotic shocks in cells of the halotolerant alga dunaliella.

The effects of osmotic shocks on polyphosphates and on the vacuolar fluorescent indicator atebrin have been investigated to test whether acidic vacuoles in the halotolerant alga Dunaliella salina have a role in osmoregulation. Upshocks and downshocks induce different patterns of polyphosphate hydrolysis. Upshocks induce rapid formation of new components, tentatively identified as 5 or 6 linear polyphosphates, formed only after upshocks with NaCl and not with glycerol, indicative of compartmentation of Na(+) into the vacuoles. Conversely, downshocks induce a slower transient accumulation of tripolyphosphates, indicating activation of a different hydrolytic process within the vacuoles. Osmotic shocks do not lead to release of atebrin from acidic vacuoles, indicating that they do not induce a major intravacuolar alkalinization. However, osmotic shocks induce transient permeability changes measured by amine-induced atebrin release from vacuoles. Hypoosmotic shocks transiently increase the permeability (up to 20-fold), whereas hyperosmotic shocks induce a rapid drop in permeability. Electron micrographs of osmotically shocked cells also reveal transient changes in the surface and internal organelles of D. salina cells. It is suggested that hyperosmotic and hypoosmotic shocks induce different changes within acidic vacuoles and in the organization and/or composition of the plasma membrane in Dunaliella.

Journal Article↗