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PubMed2002

The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase.

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.

Amino Acid Sequence↗
PubMed2002

Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C.

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.

1-Phosphatidylinositol 4-Kinase↗
PubMed1997

Effects of the amino sugars, glucosamine, mannosamine, or the fluorinated derivative 2-deoxy-fluoroglucose on the phosphatidyl inositol and glycosyl phosphatidyl inositol systems of Tetrahymena.

Sugar derivatives which have an inhibitory effect on glycosyl phosphatidyl inositol (GPI) synthesis in mammals, primarily prevented 32P incorporation in the unicellular Tetrahymena. Incorporation of 3H-mannose and 3H-inositol was induced by mannosamine. While there was a uniform effect of sugar analogues in mammals, they could not elicit totally similar effects in Tetrahymena. Analogues of sugar also affected synthesis in the phosphatidyl inositol (PI) system. Synthesis of GPI in Tetrahymena was different from that in mammals, but the metabolism of GPI and PI revealed similarities.

Animals↗
PubMed1998

Elevated [3H]inositol 1,4,5-trisphosphate binding sites and expressed inositol 1,4,5-trisphosphate receptor protein level in platelets of depressed patients.

Several reports suggest that serotonin2A (5HT2A) receptors and this receptor-mediated phosphatidyl inositol (PI) hydrolysis signal transduction system are altered in platelets of depressed patients. Inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), an important component of the PI signaling system, plays a crucial role in various physiological processes by releasing Ca2+ from intracellular stores after binding with Ins(1,4,5)P3 receptors. To examine the role of Ins(1,4,5)P3 receptors in depression, we determined [3H]Ins(1,4,5)P3 binding sites and expressed protein levels of Ins(1,4,5)P3 receptors in platelets of depressed patients (n=15) and normal control subjects (n=17). We observed that the mean Bmax of [3H]Ins(1,4,5)P3 binding to Ins(1,4,5)P3 receptors was significantly higher in platelets of depressed subjects compared with normal control subjects, whereas there was no significant difference in K(D) between these two groups. The immuno-detectable expressed level of Ins(1,4,5)P3 receptor protein was also significantly increased in depressed patients in contrast to the levels of normal control subjects. Moreover, a significant correlation was observed in Bmax and the protein level of Ins(1,4,5)P3 receptors. The increase in the number of [3H]Ins(1,4,5)P3 binding sites in platelets of depressed subjects appears to be due to an increase in the amount of Ins(1,4,5)P3 receptor proteins. These results suggest that Ins(1,4,5)P3 receptors may be involved in the pathophysiology of depression.

Adult↗
PubMed1991

Mass measurements of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in a neuronal cell line stimulated with bradykinin: inositolphosphate response shows desensitization.

In a neuronal cell line (108CC15, NG108-15) the levels of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), as measured by receptor binding assays, rise transiently after stimulation with bradykinin (EC50 approx. 150 nM). Maximal InsP3 level of 354 pmol/mg protein (15-fold basal level) is obtained at 10-15 s after addition of bradykinin, the InsP4 level rises maximally to 78 pmol/mg protein (14-fold basal level) at 20-30 s. In a rat glioma cell line, bradykinin (2 microM) causes a fast 6-fold increase in InsP3 and InsP4 levels. In the neuronal cells the bradykinin-dependent rise of the inositolphosphate levels is diminished with reduced extracellular Ca2+ concentration. However, depletion of internal Ca2+ stores does not affect the bradykinin-induced rise in InsP3 and InsP4 levels. Homologous desensitization to bradykinin occurs in the signal transduction pathway already at the production of inositolphosphates, since after a 2 min stimulation with bradykinin the rise in cellular masses of InsP3 and InsP4, inducible by a following second bradykinin stimulus, is substantially reduced.

Animals↗
PubMed1993

Effects of liposome-entrapped D-myo-inositol 1,4,5-trisphosphate and D-myo-inositol 1,3,4,5-tetrakisphosphate in the isolated rat aorta.

This study examined the effects of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)- and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4)-loaded liposomes upon the contractile activity of vascular smooth muscle, using the isolated (endothelium removed) rat aortic ring as in vitro model. While control liposomes had no effect, the administration of Ins(1,4,5)P3-containing liposomes contracted the smooth muscle preparation. Furthermore, a similar effect was seen with the administration of Ins(1,3,4,5)P4-filled liposomes but, in this case, the rings developed a significantly higher level of active tension. Pretreatment of the aortic preparation with heparin-loaded liposomes blocked the contractions induced by both Ins(1,4,5)P3- and Ins(1,3,4,5)P4-containing liposomes.

Animals↗
PubMed1991

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2(+)-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase.

The ability of D-6-deoxy-myo-inositol 1,4,5-trisphosphate [6-deoxy-Ins(1,4,5)P3], a synthetic analogue of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH-SY5Y neuroblastoma cells was investigated. 6-Deoxy-Ins(1,4,5)P3 was a full agonist (EC50 = 6.4 microM), but was some 70-fold less potent than Ins (1,4,5)P3 (EC50 = 0.09 microM), indicating that the 6-hydroxyl group of Ins(1,4,5)P3 is important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6-Deoxy-Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5-phosphatase, but inhibited both the hydrolysis of 5-[32P]+ Ins (1,4,5)P3 (Ki 76 microM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent Ki 5.7 microM). 6-Deoxy-Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins (1,4,5)P3 3-kinase.

Animals↗
PubMed1990

High-performance reversed-phase ion-pair chromatographic study of myo-inositol phosphates. Separation of myo-inositol phosphates, some common nucleotides and sugar phosphates.

A detailed study of all the major chromatographic variables affecting the retention behaviour and separation of myo-inositol phosphates in reversed-phase ion-pair chromatographic systems was carried out. The parameters studied included the eluent concentration of the pairing ion, the eluent concentration of the organic modifier and the buffer salt, the pH of the eluent, the minimum column plate count necessary for the separation of the inositol trisphosphate isomers and isocratic and gradient modes of separation. The retention behaviour of some common nucleotides and sugar phosphates was also investigated as these phosphates present chromatographic interference problems in biochemical studies based on the cellular incorporation of [32P]Pi. The separation methods developed appear to be superior to established anion-exchange separation techniques in terms of separation speed and "mildness" of the chromatographic conditions.

Chromatography, High Pressure Liquid↗
PubMed1995

Evidence for inositol tetrakisphosphate-activated Ca2+ influx pathway refilling inositol trisphosphate-sensitive Ca2+ stores in hamster eggs.

To identify the Ca2+ influx pathway responsible for maintaining Ca2+ oscillations in hamster eggs, changes in intracellular Ca2+ concentration ([Ca2+]i) were recorded using the Fura-2 fluorescent imaging technique during iontophoretic injection of inositol phosphates under voltage clamp. Both inositol 1,4,5-trisphosphate (InsP3) and 1,3,4,5-tetrakisphosphate (InsP4) caused repetitive Ca2+ transients when injected continuously into eggs, although the latter was much less effective. These Ca2+ transients were inhibited by the monoclonal antibody 18A10 to the InsP3 receptor/Ca2+ channel. In Ca(2+)-free medium, InsP4-induced Ca2+ transients were absent or much less frequent than in normal medium. A small but persistent increase in [Ca2+]i during InsP4 injection was revealed when Ca2+ uptake into InsP3-sensitive Ca2+ stores was suppressed by thapsigargin. This Ca2+ rise is due to Ca2+ entry, but not Ca2+ release, because it was: (i) increased by raising the extracellular Ca2+ concentration and abolished in Ca(2+)-free medium; (ii) larger at more negative membrane potentials which provide greater electrical driving force for Ca2+ entry; and (iii) not affected by 18A10. A moderate dose of InsP3 did not cause substantial Ca2+ entry, as tested in thapsigargin- and 18A10-treated eggs. InsP4 facilitated the restoration of Ca2+ stores after Ca2+ releases induced by pulsatile InsP3 injections. Thus, we obtained evidence for a Ca2+ influx pathway activated by InsP4 which provides Ca2+ to refill InsP3-sensitive Ca2+ stores in intact cells.

Animals↗
PubMed1992

Calcium homeostasis in Trypanosoma cruzi amastigotes: presence of inositol phosphates and lack of an inositol 1,4,5-trisphosphate-sensitive calcium pool.

The permeabilization of Trypanosoma cruzi amastigotes with digitonin allowed the study of Ca2+ fluxes between intracellular organelles in situ. In addition, fura-2 was used to determine the cytosolic Ca2+ concentration in the intact cells. When amastigotes were permeabilized in a reaction medium containing MgATP, succinate and 3.5 microM Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level, a range which correlates favorably with that detected in the intact cells with fura-2. The presence of 1 microM FCCP strongly decreased the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased and the Ca2+ set point was increased to about 0.8 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and of a large extramitochondrial Ca2+ pool, no IP3-sensitive or thapsigargin-sensitive Ca2+ release could be detected in either amastigotes or epimastigotes.

Animals↗
PubMed1991

Sequential enzymatic synthesis and biodistribution of radiolabelled inositol and inositol analogs.

The desired radiolabelled inositol (Ins) and inositol-1-phosphate (IP) were obtained from radiolabelled glucose by sequential enzyme reactions in a short time. Rapid separation and fractionation of enzymes and labelled products from the reaction mixture was achieved by HPLC with a gel-permeation chromatography column. Examination of the biodistribution of radiolabelled Ins, IP and their acetylated analogs suggested that intact Ins labelled with 11C would be more effective than 11C-labelled acetylated Ins as a brain diagnostic agent for positron emission tomographic studies of the metabolism of phosphatidylinositol and its role as a second messenger in the brain.

Acetylation↗
PubMed2000

Differential stereoselectivity of D- and L-myo-inositol 1,2,4, 5-tetrakisphosphate binding to the inositol 1,4,5-trisphosphate receptor and 3-kinase.

D- and L-myo-inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5)P(4)) were investigated for their ability to bind to the D-myo-inositol 1, 4,5-trisphosphate (Ins(1,4,5)P(3)) receptor in a bovine adrenal cortical membrane fraction, to mobilize intracellular Ca(2+) stores in Xenopus oocytes, and to bind to the rat brain Ins(1,4,5)P(3) 3-kinase overexpressed and purified in E. coli. In competitive binding experiments with the Ins(1,4,5)P(3) receptor, D-Ins(1,2,4, 5)P(4) effectively displaced [(3)H]Ins(1,4,5)P(3) in a concentration-dependent manner with a potency comparable to that of D-Ins(1,4,5)P(3), while L-Ins(1,2,4,5)P(4) was approximately 50-fold less effective than D-Ins(1,4,5)P(3) and D-Ins(1,2,4,5)P(4). The DL-Ins(1,2,4,5)P(4) racemate bound to the Ins(1,4,5)P(3) receptor with an apparent intermediate efficiency. Injection of D-Ins(1,2,4, 5)P(4) into oocytes evoked a chloride current dependent on intracellular Ca(2+) mobilization in which the agonists ranked in a similar order of potency as in the Ins(1,4,5)P(3) receptor binding. On the other hand, D-Ins(1,2,4,5)P(4) only inhibited the binding of [(3)H]Ins(1,4,5)P(3) to 3-kinase very weakly with a markedly reduced potency compared to D-Ins(1,4,5)P(3), indicating that D-Ins(1,2,4, 5)P(4) is not an effective competitor in the phosphorylation of [(3)H]-Ins(1,4,5)P(3) by 3-kinase. The results, therefore, clearly indicate that D-Ins(1,2,4,5)P(4) is as effective as D-Ins(1,4,5)P(3) in the binding to the receptor but not 3-kinase, and access of Ins(1, 2,4,5)P(4) over the Ins(1,4,5)P(3) receptor calls for stringent stereospecificity with D-Ins(1,2,4,5)P(4) being the active form in DL-Ins(1,2,4,5)P(4)-mediated Ca(2+) mobilization.

Animals↗
PubMed1993

Inositol 1,4,5-trisphosphate receptors: labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands.

We have photolabeled the inositol 1,4,5-trisphosphate (IP3) receptor and probed the IP3 ligand binding site using two novel photoaffinity ligands, [125I] (azidosalicyl)aminopropyl-IP3 ([125I]ASA-IP3) and [3H] (benzoyldihydrocinnamyl)aminopropyl-IP3 ([3H]BZDC-IP3). Both ligands have high affinity for the IP3 receptor and, when photoactivated, label the IP3 receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [125I]ASA-IP3 allowed identification of a single photolabeling site within the IP3R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [3H]BZDC-IP3 (50-60% efficiency) than with [125I]ASA-IP3 (3%), facilitating the use of [3H]BZDC-IP3 as a better ligand for the high-efficiency labeling and purification of IP3R-labeled peptides. Peptides were generated from photolabeled IP3 receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [3H]BZDC-IP3-labeled peptide, corresponding to IP3R amino acids 476-501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP3 receptor, an area suggested on the basis of mutagenesis studies to contain the IP3 recognition site.

Affinity Labels↗
PubMed1989

Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate.

1-(2-Nitrophenyl)ethyl esters of D-myo-inositol 1,4,5-trisphosphate (InsP3) have been synthesized and shown to have suitable properties for use as photolabile precursors of InsP3. Synthesis was accomplished by treatment of InsP3 with 1-(2-nitrophenyl)diazoethane in a CHCl3/water mixture. This resulted in esterification of each of the three phosphate residues in InsP3, the 1-phosphate being more reactive than the 4- or 5-phosphate. Singly esterified P-1, P-4, and P-5 esters, termed P-1, P-4, and P-5 caged InsP3, were isolated from the reaction mixture by anion-exchange HPLC and characterized by 500-MHz 1H NMR spectroscopy. Each of these caged InsP3 esters exists as a pair of diastereoisomers and was identified by examining the effects of pH and nitrophenyl ring current shielding on the chemical shifts of nonexchangeable inositol protons. 1H NMR spectra of InsP3 were analyzed for comparison. On photolysis the compounds released InsP3 with rate constants of 175 (P-1), 225 (P-4), and 280 s-1 (P-5) as determined by monitoring the aci-nitro decay reaction at pH 7.1, 0.2 M ionic strength, 21 degrees C. Quantum yields determined by steady-state near-UV photolysis were 0.65 +/- 0.08 for each compound. P-4 and P-5 caged InsP3 were the most promising biologically inactive InsP3 precursors since at concentrations up to 50 microM they did not release Ca2+ from smooth muscle sarcoplasmic reticulum (SR) and were not metabolized by vascular smooth muscle InsP3 5-phosphatase or bovine brain InsP3 3-kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Demonstration by 2H ENDOR spectroscopy that myo-inositol binds via an alkoxide bridge to the mixed-valent diiron center of myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) is a non-heme diiron oxygenase that cleaves cyclohexane-(1,2,3,5/4,6-hexa)-ol (myo-inositol, MI) to d-glucuronate. Here, we use 2H ENDOR spectroscopy to demonstrate that MI binds to the diiron(II/III) cofactor of MIOX via an alkoxide bridge, most likely involving O1. Analysis shows that MI adopts a symmetrical geometry in which the O-C-2H plane of the bridge is approximately orthogonal to the Fe-O-Fe plane.

Inositol↗
PubMed1987

Confirmation of the identities of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate by the use of one-dimensional and two-dimensional n.m.r. spectroscopy.

Multinuclear n.m.r. spectroscopy, including the use of two-dimensional methodology, was used to confirm the identity of inositol 1,3,4-triphosphate and its metabolic precursor inositol 1,3,4,5-tetrakisphosphate. The cyclohexane ring in each molecule exhibits a chair conformation with all phosphate groups occupying equatorial positions.

Inositol 1,4,5-Trisphosphate↗
PubMed1988

Metabolism of inositol 1,3,4,5-tetrakisphosphate by human erythrocyte membranes. A new mechanism for the formation of inositol 1,4,5-trisphosphate.

Human erythrocyte membranes metabolize inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] in the presence of Mg2+. In the absence of Mg2+ a less rapid conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3 was revealed. Such an enzyme activity, if present in hormonally sensitive cells, could provide a mechanism for maintaining constant concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, important for stimulation of Ca2+ entry after Ca2+ mobilization.

Erythrocyte Membrane↗