Rat liver contains a potent endogenous inhibitor of inositol 1,3,4,5-tetrakisphosphate 3-phosphatase.
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ABSTRACT:
SUBMITTER: Hodgson ME
PROVIDER: S-EPMC1131373 | biostudies-other | 1990 May
REPOSITORIES: biostudies-other
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Project description:Many neutrophil functions are regulated by phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to PtdIns(3,4,5)P3. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,3,4,5)P4 concentration. Depletion of Ins(1,3,4,5)P4 by deleting the gene encoding InsP3KB, which converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4, enhanced membrane translocation of the PtdIns(3,4,5)P3-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,3,4,5)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,3,4,5)P4 in cellular signal transduction pathways and provide another mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.
| S-EPMC2084373 | biostudies-literature
Project description:The influence of highly phosphorylated inositol phosphates on the Ins(1,3,4,5)P4 3-phosphatase enriched from the soluble fraction of pig brain was tested, using [5-32P]Ins(1,3,4,5)P4 as substrate. Both Ins(1,3,4,5,6)P5 and InsP6 were very potent inhibitors of the Ins(1,3,4,5)P4 3-phosphatase. The Ki values were approximately 60 nM and approximately 3 nM for Ins(1,3,4,5,6)P5 and InsP6 respectively. Ins(1,3,4,5,6)P5 and InsP6 also inhibited the Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. Using Ins(1,3,4,5)P4 as substrate, the Ki values were about 35 microM and 15 microM for Ins(1,3,4,5,6)P5 and InsP6 respectively. The concentrations which led to a 50% inhibition of Ins(1,4,5)P3 (0.5 microM) degradation by the 5-phosphatase were about 20 and 10 microM for the pentakis- and hexakis-phosphate respectively. As the intracellular concentrations of Ins(1,3,4,5,6)P5 and InsP6 are high (up to 60 microM) compared with those of the inositol trisphosphates and tetrakisphosphates, it is possible that the highly phosphorylated inositol phosphates act as regulators in the metabolism of Ca(2+)-mobilizing inositol phosphates.
| S-EPMC1151471 | biostudies-other
Project description:Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] analogues were synthesized and their effects on [3H]Ins(1,3,4,5)P4 5-phosphatase, [3H]Ins(1,3,4,5)P4 3-phosphatase and [3H]inositol 1,4,5-trisphosphate [3H]Ins(1,4,5)P3] 5-phosphatase activities were examined. The Ins(1,3,4,5)P4 analogue with the aminobenzoyl group at the 2-position of Ins(1,3,4,5)P4 inhibited the hydrolysis of 5-phosphate of [3H]Ins(1,3,4,5)P4 catalysed by erythrocyte ghosts, with a lower Ki value than seen with Ins(1,3,4,5)P4, whereas the analogue with the aminocyclohexanecarbonyl group at the same position had a higher Ki value. The Ins(1,4,5)P3 analogues that we had previously synthesized were also capable of inhibiting this process, with the same tendency as Ins(1,3,4,5)P4 analogues. Such differences in the potency among Ins(1,3,4,5)P4 and Ins(1,4,5)P3 analogues were applicable to other phosphatase activities, namely [3H]Ins(1,3,4,5)P4 3-phosphatase and [3H]Ins(1,4,5)P3 5-phosphatase. These results suggest that the active sites of these enzymes may catalyse the dephosphorylation in a similar fashion.
| S-EPMC1151095 | biostudies-other
Project description:1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.
| S-EPMC1148250 | biostudies-other
Project description:We have previously shown that Ins(1,3,4,5)P4 is degraded to Ins(1,4,5)P3 by a soluble Ins(1,3,4,5)P4 3-phosphatase from pig brain [Höer, Kwiatkowski, Seib, Rosenthal, Schultz & Oberdisse (1988) Biochem. Biophys. Res. Commun. 154, 668-675]. Here we present some properties of this enzyme using [5-32P]Ins(1,3,4,5)P4 as substrate. The molecular mass, estimated by gel filtration chromatography on a Superose 6 column, was determined to be 36 kDa. The 3-phosphatase showed a high affinity towards the substrate Ins(1,3,4,5)P4 (Km approximately 400 nM); the Vmax. of the freshly prepared enzyme was 2 nmol/min per mg of protein. The influence of Ins(1,4,5)P3 and Ins(1,3,4)P3, the reaction products of Ins(1,3,4,5)P4 hydrolysis by either 3- or 5-phosphatase respectively, on the 3-phosphatase was tested. Both isomers inhibited the enzyme, with Ki values of about 2 microM and 1.75 microM for Ins(1,3,4)P3 and Ins(1,4,5)P3 respectively. Enzyme activity was not influenced by Mg2+ up to 30 mM or Ca2+ up to 1 mM. Commercially available Ins(3,4,5,6)P4 from turkey erythrocytes produced a marked inhibition of the 3-phosphatase (Ki approximately 500 nM). Significant inhibitory effects on enzyme activity were also found with GTP and the pyrimidine nucleotides UTP and CTP. The kinetic data presented here suggest that the Ins(1,3,4,5)P4 3-phosphatase may be regulated by the intracellular concentrations of inositol tris- and tetrakis-phosphates.
| S-EPMC1131790 | biostudies-other
Project description:Previous studies have shown that most of the inositol 1,4,5-trisphosphate/inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity of rat hepatocytes is associated with the plasma membrane [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. We now show that the specific activity of this enzyme is highest in the bile-canalicular domain of the plasma membrane, at the opposite pole of the hepatocyte from the presumed site of receptor-mediated formation of inositol 1,4,5-trisphosphate. In intact hepatocytes and in sealed membrane vesicles originating from the bile-canalicular domain of the plasma membrane, the 5-phosphatase activity was mostly latent and therefore located at the cytoplasmic surface. A substantial amount of 5-phosphatase was also found in rat liver endosomal fractions, particularly a 'late' endosomal subfraction, indicating that this enzyme may be transported between the sinusoidal plasma membrane and other cellular membranes.
| S-EPMC1135418 | biostudies-other
Project description:1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude 'P2' cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.
| S-EPMC1150196 | biostudies-other
Project description:Dephosphorylation of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was measured in both the soluble and the particulate fractions of rat brain homogenates. Analysis of the hydrolysis of [4,5-32P]Ins(1,3,4,5)P4 showed that for both fractions the 5-phosphate of Ins(1,3,4,5)P4 was removed and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] was specifically produced. In the soluble fraction, Ins(1,3,4)P3 was further hydrolysed at the 1-phosphate position to inositol 3,4-bisphosphate[Ins(3,4)P2]. DEAE-cellulose chromatography of the soluble fraction separated the phosphatase activities into three peaks. The first hydrolysed both Ins(1,3,4,5)P4 and inositol 1,4,5-trisphosphate, the second inositol 1-phosphate and the third Ins(1,3,4)P3 and inositol 1,4-bisphosphate, [Ins(1,4)P2]. Further purification of the third peak on either Sephacryl S-200 or Blue Sepharose could not dissociate these two activities [i.e. with Ins(1,4)P2 and Ins(1,3,4)P3 as substrates]. The dephosphorylation of Ins(1,3,4)P3 could be inhibited by the addition of Li+.
| S-EPMC1148459 | biostudies-other
Project description:We have previously found that for permeabilized L1210 cells, low micromolar concentrations of Ins(1,3,4,5)P4 added prior to Ins(2,4,5)P3 enhance the effects of suboptimal concentrations of Ins(2,4,5)P3 in causing Ca2+ release from InsP3-sensitive Ca2+ stores [Cullen, Irvine and Dawson (1990) Biochem J. 271, 549-553]. If this was due either to some conversion of added Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by the 3-phosphatase, or to Ins(1,3,4,5)P4 acting as a weak (or partial) agonist on the InsP3 receptor it would be expected that,in the presence of thimerosal to sensitize the InsP3 receptor, the dose-response curve to Ins(1,3,4,5)P4 would be left-shifted by the same extent as that of Ins(1,4,5)P3. This was found not to be the case; the dose-response curve to Ins(1,3,4,5)P4 was not shifted at all by thimerosal. Furthermore, L-Ins(1,3,4,5)P4, which can displace radiolabelled D-Ins(1,3,4,5)P4 but not D-Ins(1,4,5)P3 from their respective high-affinity binding sites, mimicked the effects of D-Ins(1,3,4,5)P4 in enhancing the slow phase of Ins(2,4,5)P3-stimulated Ca2+ release. Ins(1,3,4,5)P4 caused an increase in magnitude of the slow phase of InsP3-stimulated Ca2+ release leaving the magnitude of the fast phase unaltered, in contrast to increasing Ins(2,4,5)P3 concentrations which increased the size of both phases. In addition, Ins(1,3,4,5)P4 decreased the rate constant for the slow phase of Ca2+ release. These findings point strongly to the conclusion that InsP4 is not working directly via the InsP3 receptor but indirectly via an InsP4 receptor.
| S-EPMC1217129 | biostudies-other
Project description:Carbachol stimulation of muscarinic receptors in rat cortical slices prelabelled with myo-[2-3H]inositol caused the rapid formation of a novel inositol polyphosphate. Evidence derived from its chromatographic behaviour, and from the structure of the products formed in partial dephosphorylation experiments, suggests that it is probably D-myo-inositol 1,3,4,5-tetrakisphosphate. An enzyme in human red cell membranes specifically removes the 5-phosphate from it to form inositol 1,3,4-trisphosphate. It is suggested that inositol 1,3,4,5-tetrakisphosphate is likely to be a second messenger, and that it is the precursor of inositol 1,3,4-trisphosphate and possibly of inositol 1,4,5-trisphosphate.
| S-EPMC1152860 | biostudies-other