The norepinephrine-stimulated inositol phosphate response in human atria

Inositol phosphate release and metabolism were studied in right atrial appendages obtained from 18 patients undergoing coronary artery bypass surgery and/or mitral valve replacement. [3H]Inositol-labeled human atria contained inositol(1,4. 5)trisphosphate, inositol(1,4)bisphosphate and the 1- (or 3) and 4-isomers of inositol monophosphate. Addition of norepinephrine (100 mumol/l) activated the release of inositol phosphates, as indicated by increased [3H]inositol label in all of these inositol phosphates. However, the phosphorylation product of inositol (1.4.5)trisphosphate, inositol-(1,3,4,5)tetrakisphosphate, and its metabolic products were not detected, either in control or stimulated atria. Similar inositol phosphate profiles were observed in rat right atria. Furthermore, both human and rat atria contained high concentrations of inositol(1,4,5)trisphosphate, which were not observed to increase with norepinephrine stimulation. The inositol phosphate responses to norepinephrine in rat and human cardiac tissue appear to be similar, except for the generally lower activity observed in human tissue. Thus, the rat provides a suitable model for the study of cardiac phosphatidylinositol turnover.     

Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.     

SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase

Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by Salmonella dublin, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro. SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca2+-dependent chloride secretion. In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate to yield inositol 1,4,5, 6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the inhibition of chloride secretion by phosphatidylinositol 3,4,5-trisphosphate [Eckmann, L., Rudolf, M. T., Ptasznik, A., Schultz, C., Jiang, T., Wolfson, N., Tsien, R., Fierer, J., Shears, S. B., Kagnoff, M. F., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14456-14460]. Mutation of a conserved cysteine that abolishes phosphatase activity of SopB results in a mutant strain, S. dublin SB c/s, with decreased ability to induce fluid secretion in infected calf intestine loops. Moreover, HeLa cells infected with S. dublin SB c/s do not accumulate high levels of inositol 1,4,5,6-tetrakisphosphate that are characteristic of wild-type S. dublin-infected cells. Therefore, SopB mediates virulence by interdicting inositol phosphate signaling pathways.     

Hydrolysis of phosphatidylinositol 3,4-bisphosphate by inositol polyphosphate 4-phosphatase isolated by affinity elution chromatography

Inositol polyphosphate 4-phosphatase is a monomeric 110-kDa protein that hydrolyzes two substrates in the inositol phosphate pathway. Inositol 3,4-bisphosphate is converted to inositol 3-phosphate, and inositol 1,3,4-trisphosphate is converted to inositol 1,3-bisphosphate. We have exploited the fact that inositol hexasulfate inhibits the enzyme to devise an affinity elution scheme from a Mono S cation exchange column that resulted in an 11,300-fold purified preparation of rat brain 4-phosphatase. The resulting 4-phosphatase hydrolyzed phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3-phosphate with a first order rate constant 120-fold greater than that for inositol 3,4-bisphosphate and 900-fold greater than that for inositol 1,3,4-trisphosphate. This is now the third example wherein the same enzyme hydrolyzes both an inositol lipid and its analogous inositol phosphate.     

Lack of correlation between apoptosis and DNA single-strand breaks in X-irradiated human peripheral blood mononuclear cells in the course of ageing

Inositol(1,3,4,5)tetrakisphosphate (InsP4) and phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42IP4 previously purified from pig cerebellum, which binds InsP4 (Kd approximately 2 nM) and PtdInsP3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42IP4 (pig) is highly homologous to centaurin-alpha, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42IP4 is expressed in rat brain and how it might be related to centaurin-alpha. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42IP4, but not to centaurin-alpha. The existence of p42IP4 in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-alpha-like protein in rat brain; and (3) the amino acid sequences deduced from p42IP4 cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42IP4 from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.     

Effect of pertussis toxin and B-oligomer on platelet-activating factor-induced generation of inositol phosphates in porcine alveolar macrophages

OBJECTIVE: To test the hypothesis that platelet-activating factor (PAF) induces inositol phosphate turnover through a receptor-linked, pertussis toxin-sensitive guanine nucleotide-binding (G) protein-dependent pathway in porcine alveolar macrophages. DESIGN: Randomized complete block design was used with 2 or 3 replicates/block. ANIMALS: Porcine alveolar macrophages were obtained by lavage of excised lungs from Yorkshire-type pigs (mean +/- SEM, 21 +/- 2 kg). PROCEDURE: Phospholipase C activation was assessed, using anion exchange chromatography to measure accumulation of inositol phosphates in [3H]myo-inositol-labeled alveolar macrophages. Macrophages were incubated with saline solution, pertussis toxin (4.75 nM), or B-oligomer (4.75 nM) for 2 hours. Cells then were washed and incubated for 5 minutes with PAF (0, 0.1, 1.0, or 10 microM; n = 15). Results were expressed as total inositol phosphates (inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate). RESULTS: Concentrations of total inositol phosphates were significantly (P < 0.05) increased to 162 +/- 7, 172 +/- 4, and 194 +/- 9% of control in response to 0.1, 1.0, and 10 microM PAF, respectively. Pertussis toxin attenuated the PAF-induced increase in total inositol phosphates by approximately 50% (P < 0.05). The B-oligomer of pertussis toxin failed to modify PAF-induced increases in total inositol phosphates. The specific PAF receptor antagonist WEB 2086 markedly attenuated PAF-induced. (10 microM) increase in inositol phosphates. CONCLUSIONS: We conclude that PAF stimulates accumulation of inositol phosphates through a specific receptor and that a pertussis toxin-sensitive G protein is involved in the signal transduction process leading to activation of phospholipase C in porcine alveolar macrophages.     

The diversity and possible functions of the inositol polyphosphate 5-phosphatases

Distinct forms of inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates, i.e., inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), inositol 1,3,4, 5-tetrakisphosphate (Ins(1,3,4,5)P4), phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) or phosphatidylinositol 3,4, 5-trisphosphate (PtdIns(3,4,5)P3). In mammalian cells, this family contains a series of distinct genes and splice variants. All inositol polyphosphate 5-phosphatases share a 5-phosphatase domain and various protein modules probably responsible for specific cell localisation or recruitment (SH2 domain, proline-rich sequences, prenylation sites, etc.). Type I Ins(1,4,5)P3 5-phosphatase also uses Ins(1,3,4,5)P4 but not the phosphoinositides as substrates. This enzyme is targeted to specific membranes by means of a prenylation site. Type II 5-phosphatases can use both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 as substrates. Five mammalian enzymes and multiple splice variants are known: INPP5P or inositol polyphosphate 5-phosphatase II, OCRL (a Golgi protein implicated in the Lowe oculocerebrorenal syndrome), synaptojanin (a protein involved in the recycling of synaptic vesicles), SHIP 1 and SHIP 2 (or SH2-containing inositol 5-phosphatases). As discussed in this review, the substrate specificity, regulatory mechanisms, subcellular localisation and tissue specificity indicate that the different 5-phosphatase isoforms may play specific roles. As known in the dephosphorylation of tyrosine containing substrates by the tyrosine protein phosphatases or in the metabolism of cyclic nucleotides by the cyclic nucleotide phosphodiesterases, inositol polyphosphate 5-phosphatases directly participate in the control of second messengers in response to both activation or inhibitory cell signalling.     

Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells

1. The effects of extracellular adenosine 5'-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5'-triphosphate (2MeSATP) and uridine 5'-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.     

Production of recombinant human brain type I inositol-1,4,5-trisphosphate 5-phosphatase in Escherichia coli. Lack of phosphorylation by protein kinase C

The dephosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,4-bisphosphate is catalyzed by InsP3 5-phosphatase. The coding region of human brain type I InsP3 5-phosphatase was expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The relative molecular mass of the purified fusion protein (MBP-InsP3-5-phosphatase) was approximately M(r) 85,000 as analysed by SDS/PAGE. The yield was about 10 mg fusion protein/l lysate. After cleavage from MBP with factor Xa, the specific activity of recombinant 5-phosphatase was 120-250 mumol.mg-1.min-1. The molecular mass of purified protein by SDS/PAGE was M(r) 43,000. The activity was inactivated by p-hydroxymercuribenzoate. The possibility that protein kinase C might phosphorylate InsP3 5-phosphatase was tested on the purified 43,000 M(r) protein. In this study, we show that recombinant 5-phosphatase is not a substrate of protein kinase C.     

Biotransformation of 4-dimethylaminophenol: reaction with glutathione, and some properties of the reaction products

4-Dimethylaminophenol (DMAP) forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. In solutions of purified human hemoglobin, quick binding of oxidized DMAP to the globin moiety of hemoglobin terminates this reaction. Reduced glutathione in high concentrations, as in the red cell, substantially diminished binding of oxidized DMAP to hemoglobin by formation of S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-tris-glutathione (tris-(GS)-DMAP), which does not form ferrihemoglobin. In the presence of reduced glutathione, DMAP disappeared more rapidly from hemoglobin solutions than in its absence. The formation of tris(GS)-DMAP in red cells was found to be of importance for the termination of catalytic ferrihemoglobin formation by DMAP in vivo. With low concentrations of GSH, DMAP in hemoglobin solutions formed another conjugate, (GS)-DMAP, S,S(2-dimethylamino-5-hydroxy-1,3-phenylene)-bis-glutathione. Similar to DMAP, bis(GS)-DMAP catalyzed the formation of ferrihemoglobin. As the oxidized bis(GS)-DMAP was bound to hemoglobin more slowly and to a lesser extent, it produced more ferrihemoglobin than DMAP. In contrast to the reactions of DMAP with hemoglobin, hydrogen peroxide and superoxide radicals are involved in the ferrihemoglobin formation by bis(GS)-DMAP. The radicals accelerate the oxidation of bis(GS)-DMAP and thereby the ferrihemoglobin formation.     

A bifunctional enzyme catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Purification and characterization of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

Biosynthesis of N-acetylneuraminic acid (Neu5Ac), a prominent component of glycoconjugates, is initiated by the action of UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1. 3.14) and N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60). We demonstrate for the first time that the two activities are parts of one bifunctional enzyme in rat liver. The enzyme was purified to homogeneity from rat liver cytosol using salmine sulfate precipitation and chromatography on phenyl-Sepharose, ATP-agarose, and Mono Q. The purification resulted in one polypeptide with an apparent molecular mass of 75 kDa. Immunoprecipitation with a polyclonal antibody against the polypeptide reduced both enzyme activities in equal amounts. Gel filtration analysis of purified UDP-GlcNAc 2-epimerase/ManNAc kinase showed that the polypeptide self-associates as a dimer and as a hexamer with apparent molecular masses of 150 and 450 kDa, respectively. The hexamer was fully active for both enzyme activities, whereas the dimer catalyzed only the phosphorylation of N-acetylmannosamine (ManNAc). Incubation of the dimer with UDP-N-acetylglucosamine led to reassembly of the fully active hexamer; maximal quantities of the hexamer were produced after incubation for 3 h. Kinetic analysis of purified hexameric and dimeric enzyme revealed significantly lower Michaelis constants (93 +/- 3 to 121 +/- 15 microM for ManNAc and 1.18 +/- 0. 13 to 1.67 +/- 0.20 mM for ATP) and higher cooperativity (Hill coefficients of 1.42 +/- 0.16 to 1.17 +/- 0.06 for ManNAc and 1.30 +/- 0.09 to 1.05 +/- 0.14 for ATP) for the hexamer for both substrates of ManNAc kinase. The Michaelis constant of UDP-GlcNAc 2-epimerase for its substrate was 11 +/- 2 microM. The Hill coefficient of 0.45 +/- 0.07 represents strongly negative cooperativity in substrate binding. UDP-GlcNAc 2-epimerase was feedback inhibited by CMP-Neu5Ac. Complete inhibition was achieved with 60 microM CMP-Neu5Ac, and highly positive cooperativity (Hill coefficient of 4.1) was found for inhibitor binding.     

cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.     

Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.     

Isolation and characterization of a novel inositol hexakisphosphate binding protein from mammalian cell cytosol

Inositol hexakisphosphate (IP6) is present in most mammalian cells, although its intracellular function is as yet undefined. We find that the total protein fraction from bovine brain cytosol contains a significant level of specific binding for IP6 precipitable with 40% saturated ammonium sulfate. A protein complex has been isolated from this fraction that specifically binds IP6 and is purified about 500-fold over the cytosol. The IP6 binding protein (IP6BP) chromatographs as a single peak of binding activity on a gel exclusion column, with a Stokes radius equivalent to 266 +/- 14 kDa. The IP6BP is a heterooligomeric complex composed of a number of subunits with molecular weights varying from 23,000 to 60,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Scatchard analyses of IP6 binding of both the crude ammonium sulfate fraction and the purified complex show the presence of a similar high-affinity binding site (Kd approximately 6.0 nM). Bmax for the purified fraction is 1.8 nmol of IP6/mg of protein or 0.48 mol of IP6 bound/mol of complex. Other inositol polyphosphates, such as inositol 1,3,4,5,6-pentakisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate, are poor competitors for IP6 binding to the purified complex. The purification scheme, when applied to a rat liver cytosol fraction, yields a similar IP6BP. This complex has an apparent size of 512,000 using gel exclusion chromatography and contains an additional protein band with M(r) = 97,000 by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biological relevance of the binding of calcium ions by inositol phosphates

The binding of Ca2+ (chelation) by myo-inositol polyphosphates at pH 7.0 was studied using a Ca(2+)-sensitive electrode. Glucose 6-phosphate (used as a model for a monophosphate) bound Ca2+ with an affinity of 152 +/- 31 liters/mol and a molar ratio of 0.94 +/- 0.02. Inositol 3,4-bisphosphate, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol hexakisphosphate showed affinities of 9.0 +/- 2.1 x 10(3), 6.3 +/- 1.5 x 10(3), 6.2 x 10(4), and 1.92 +/- 0.47 x 10(5) liters/mol, respectively, and molar ratios of 0.92 +/- 0.49, 0.95 +/- 0.10, 0.75, and 2.5 +/- 0.5. In general, the affinity increased with the number of phosphate substituents on the inositol ring, although the stereochemistry is also expected to be important. This suggests that for the physiologically relevant inositol phosphates (tris-, tetrakis-, pentakis-, and hexakis-) half-maximal Ca2+ binding will occur in the Ca2+ concentration range of approximately 5 x 10(-6) to 2 x 10(-4) M. This range lies between the basal intracellular and the fee extracellular Ca2+ levels (10(-7) and 10(-3) M), respectively, and may therefore be of physiological importance. Chelation provides a possible simple explanation for the inhibition by Ca2+ of inositol 1,4,5-trisphosphate binding to its receptor in rat cerebellum and other tissues. It may also have a role in limiting inositol phosphate-mediated increases in intracellular Ca2+.     

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.     

Differential regulation of inositol 1,4,5-trisphosphate by co-existing P2Y-purinoceptors and nucleotide receptors on bovine aortic endothelial cells

1. We have examined the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) responses in bovine aortic endothelial (BAE) cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. Exchange of medium on BAE cells in the absence of agonist was found to be a stimulus for Ins(1,4,5)P3 generation. BAE cells stimulated with 100 microM ATP, 30 microM 2MeSATP (an agonist at P2Y-purinoceptors but not nucleotide receptors) or 100 microM UTP (an agonist at nucleotide receptors but not P2Y-purinoceptors) gave Ins(1,4,5)P3 responses above that caused by exchange of medium. The time course was rapid, with peak response within the first 5 s and levels returning close to basal after 30 s of stimulation. 3. Significant differences in Ins(1,4,5)P3 responses to 100 microM UTP and 30 microM 2MeSATP stimulation were observed. The response to UTP was reproducibly more sustained than that to 2MeSATP. 4. Stimulation of BAE cells with 100 microM UTP plus 30 microM 2MeSATP produced a response statistically indistinguishable from that predicted by addition of the responses to the two agonists in isolation. 5. The Ins(1,4,5)P3 response to UTP was attenuated to 25% of control by pretreatment of BAE cells with pertussis toxin. Responses to 2MeSATP and ADP were essentially unaffected. ATP stimulation was reduced to 65% of control. 6. Activation of protein kinase C with tetradecanoyl phorbol acetate (TPA) profoundly inhibited Ins(1,4,5)P3 responses to 2MeSATP and ADP but had no effect on UTP stimulation. The protein kinase C inhibitor, Ro 31-8220, enhanced responses to 2MeSATP, ADP and ATP but no effect was observed on UTP stimulation. 7. These observations show that nucleotide and P2Y-receptors mobilise the second messenger Ins(1,4,5)P3 by separate routes resulting in different patterns of generation and suggest that while ATP activates both receptors, ADP principally influences these cells by interacting with the P2Y-purinoceptors.     

Phosphorylation of glycerol and dihydroxyacetone in Acetobacter xylinum and its possible regulatory role

Extracts of Acetobacter xylinum catalyze the phosphorylation of glycerol and dihydroxyacetone (DHA) by adenosine 5'-triphosphate (ATP) to form, respectively, L-alpha-glycerophosphate and DHA phosphate. The ability to promote phosphorylation of glycerol and DHA was higher in glycerol-grown cells than in glucose- or succinate-grown cells. The activity of glycerol kinase in extracts is compatible with the overall rate of glycerol oxidation in vivo. The glycerol-DHA kinase has been purified 210-fold from extracts, and its molecular weight was determined to be 50,000 by gel filtration. The glycerol kinase to DHA kinase activity ratio remained essentially constant at 1.6 at all stages of purification. The optimal pH for both reactions was 8.4 to 9.2. Reaction rates with the purified enzyme were hyperbolic functions of glycerol, DHA, and ATP. The Km for glycerol is 0.5 mM and that for DHA is 5 mM; both are independent of the ATP concentration. The Km for ATP in both kinase reactions is 0.5 mM and is independent of glycerol and DHA concentrations. Glycerol and DHA are competitive substrates with Ki values equal to their respective Km values as substrates. D-Glyceraldehyde and l-Glyceraldehyde were not phosphorylated and did not inhibit the enzyme. Among the nucleotide triphosphates tested, only ATP was active as the phosphoryl group donor. Fructose diphosphate (FDP) inhibited both kinase activities competitively with respect to ATP (Ki= 0.02 mM) and noncompetitively with respect to glycerol and DHA. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) inhibited both enzymic activities competitively with respect to ATP (Ki (ADP) = 0.4 mM; Ki (AMP) =0.25 mM). A. xylinum cells with a high FDP content did not grow on glycerol. Depletion of cellular FDP by starvation enabled rapid growth on glycerol. It is concluded that a single enzyme from A. xylinum is responsible for the phosphorylation of both glycerol and DHA. This as well as the sensitivity of the enzyme to inhibition by FDP and AMP suggest that it has a regulatory role in glycerol metabolism.     

Decreased ATP-induced synthesis and Ca(2+)-stimulated degradation of polyphosphoinositides in pancreatic islets from neonatally streptozotocin-diabetic rats

Phosphoinositide (PI) synthesis and hydrolysis were investigated in pancreatic islet homogenates from neonatal streptozotocin diabetic (n-STZ) and control rats. In the diabetics, ATP, in absence of Ca2+, failed to increase the amount of phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidyl inositol 4, 5-bisphosphate (PtdInsP2) at variance with the pattern in controls. Also, the Ca(2+)-stimulated generation of inositol phosphates (InsP) was dramatically decreased, whether in the absence or presence of ATP. Moreover, phosphatidylinositol (PtdIns) kinase activity was reduced while PtdInsP kinase activity was not impaired. These data suggest that the suppressed formation of PtdInsP and subsequent PtdInsP2 synthesis, concomitantly with a decreased Ca(2+)-stimulated phospholipase C activity, may participate to the alteration of the PI pathway, the limitation of the InsP production, and finally the impairment of the insulin release in the n-STZ model of non-insulin-dependent diabetes.