Enzymic dephosphorylation of D-myo-inositol 1,4-bisphosphate in rat brain.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] phosphatase activities were measured in both 180,000 g (60 min) particulate and supernatant fractions of rat brain homogenates. Although Ins(1,4,5)P3 was mostly hydrolysed by a particulate phosphatase [Erneux, Delvaux, Moreau & Dumont (1986) Biochem. Biophys. Res. Commun. 134, 351-358], Ins(1,4)P2 phosphatase was predominantly soluble. The latter enzyme was Mg2+-dependent and sensitive to thiol-blocking agents (e.g. p-hydroxymercuribenzoate). In contrast with Ins(1,4,5)P3 phosphatase activity measured in the soluble fraction, Ins(1,4)P2 phosphatase was insensitive to 0.001-1 mM-2,3-bisphosphoglycerate. Lithium salts, widely used in psychiatric treatment, inhibited both Ins(1,4)P2 and Ins(1)P1 phosphatase activities of the crude soluble fraction. In particular, 50% inhibition of phosphatase activity, with 2 microM-Ins(1,4)P2 as substrate, was achieved at 3-5 mM-LiCl. At these concentrations, LiCl did not change Ins(1,4,5)P3 phosphatase activity measured in the same fraction with 1-4 microM-Ins(1,4,5)P3 as substrate. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved three phosphatase activities. These forms, peaks I, II and III, dephosphorylated Ins(1,4,5)P3, Ins(1)P1 and Ins(1,4)P2 respectively. If LiCl (10 mM) was included in the assay mixture, it inhibited both peak-II Ins(1)P1 phosphatase and peak-III Ins(1,4)P2 phosphatase, suggesting the existence of at least two Li+-sensitive phosphatases.     

Metabolism of D-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate.

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.     

Preferential localization of rat liver D-myo-inositol 1,4,5-trisphosphate/1,3,4,5-tetrakisphosphate 5-phosphatase in bile-canalicular plasma membrane and ''late'' endosomal vesicles.

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.     

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a ''PB-like'' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.     

Demonstration of inositol 1,3,4,5-tetrakisphosphate receptor binding

Inositol 1,3,4,5-tetrakisphosphate (InsP4) is produced rapidly upon stimulation of the phosphoinositide system and may serve as a second messenger in hormone and neurotransmitter action. In this report we demonstrate specific binding sites for [3H]InsP4 in rat tissue membranes. In cerebellar membranes, [3H]InsP4 binding sites are displaced both by InsP4 and inositol 1,4,5-trisphosphate (InsP3) with similar potency (IC50 approximately equal to 300 nM) whereas several other inositol phosphates are much weaker. We have distinguished the InsP4 binding site from the InsP3 receptor binding site by differences in brain regional and tissue distribution, affinity for InsP4 and InsP3, and sensitivity to calcium.     

D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate, a mimic of d-myo-inositol 1,3,4,5-tetrakisphosphate: biological activity and pH-dependent conformational properties

D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate [D-6-deoxy-Ins(1,3,4,5)P(4)] 3 is a novel deoxygenated analogue of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)] 2, a central and enigmatic molecule in the polyphosphoinositide pathway of cellular signalling. D-6-Deoxy-Ins(1,3,4,5)P(4) is a moderate inhibitor of Ins(1,4,5)P(3) 5-phosphatase [1.8microM] compared to Ins(1,3,4,5)P(4) [0.15microM] and similar to that of L-Ins(1,3,4,5)P(4) [1.8microM]. In displacement of [(3)H] Ins(1,4,5)P(3) from the rat cerebellar Ins(1,4,5)P(3) receptor, while slightly weaker [IC(50)=800nM] than that of D-Ins(1,3,4,5)P(4) [IC(50)=220nM], 3 is less markedly different and again similar to that of L-Ins(1,3,4,5)P(4) [IC(50)=660nM]. 3 is an activator of I(CRAC) when inward currents are measured in RBL-2H3-M1 cells using patch-clamp electrophysiological techniques with a facilitation curve different to that of Ins(1,3,4,5)P(4). Physicochemical properties were studied by potentiometric (31)P and (1)H NMR titrations and were similar to those of Ins(1,3,4,5)P(4) apart from the observation of a biphasic titration curve for the P1 phosphate group. A novel vicinal phosphate charge-induced conformational change of the inositol ring above pH 10 was observed for D-6-deoxy-Ins(1,3,4,5)P(4) that would normally be hindered because of the central stabilising role played by the 6-OH group in Ins(1,3,4,5)P(4). We conclude that the 6-OH group in Ins(1,3,4,5)P(4) is crucial for its physicochemical behaviour and biological properties of this key inositol phosphate.     

Rapid formation of inositol 1,3,4,5-tetrakisphosphate following muscarinic receptor stimulation of rat cerebral cortical slices.

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.     

Regulation of innate immunity by inositol 1,3,4,5-tetrakisphosphate

Many neutrophil functions are mediated by PtdIns(3,4,5)P3 that exerts its role by mediating protein translocation via binding to their PH-domains. Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) binds the same PH domain, competes for its binding to PtdIns(3,4,5)P3, and thus negatively regulates PtdIns(3,4,5)P3 signaling. In neutrophils, chemoattractant stimulation triggers rapid elevation in Ins(1,3,4,5)P4 level. Depletion of Ins(1,3,4,5)P4 by deleting InsP3KB, the major enzyme producing Ins(1,3,4,5)P4 in neutrophils, augments PtdIns(3,4,5)P3 downstream signals, leading to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. InsP3KB gene is also expressed in hematopoietic stem/progenitor cells. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population is expanded and the proliferation of GMP cells is accelerated. As results, neutrophil production in the bone marrow is enhanced and peripheral blood neutrophil count is elevated. Ins(1,3,4,5)P4 also plays a role in maintaining neutrophil survival. Depletion of Ins(1,3,4,5)P4 leads to accelerated neutrophil spontaneous death. Finally, InsP3KB and Ins(1,3,4,5)P4 are essential components in bacterial killing by neutrophils. Despite of the augmented neutrophil recruitment, the clearance of bacteria in the InsP3KB knockout mice is significantly impaired. Collectively, these findings establish InsP3KB and its product Ins(1,3,4,5)P4 as essential modulators of neutrophil function and innate immunity.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

Agonist-induced calcium signaling is impaired in fibroblasts overproducing inositol 1,3,4,5-tetrakisphosphate.

The proposed Ca(2+)-signaling actions of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), formed by phosphorylation of the primary Ca(2+)-mobilizing messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), were analyzed in NIH 3T3 and CCL39 fibroblasts transfected with rat brain Ins(1,4,5)P3 3-kinase. In such kinase-transfected cells, the conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P4 during agonist stimulation was greatly increased, with a concomitant reduction in Ins(1,4,5)P3 levels and attenuation of both the cytoplasmic Ca2+ increase and the Ca2+ influx response. This reduction in Ca2+ signaling was observed during activation of receptors coupled to guanine nucleotide-binding proteins (thrombin and bradykinin), as well as with those possessing tyrosine kinase activity. Single-cell Ca2+ measurements in CCL39 cells revealed that the smaller averaged Ca2+ response of enzyme-transfected cells was due to a marked increase in the number of cells expressing small and slow Ca2+ increases, in contrast to the predominantly large and rapid Ca2+ responses of vector-transfected controls. There was no evidence that high Ins(1,3,4,5)P4 levels promote Ca2+ mobilization, Ca2+ entry, or Ca2+ sequestration. These data indicate that Ins(1,4,5)P3 is the major determinant of the agonist-induced Ca2+ signal in fibroblasts and that Ins(1,3,4,5)P4 does not appear to contribute significantly to this process. Instead, Ins(1,4,5)P3 3-kinase may serve as a negative regulator of the Ca(2+)-phosphoinositide signal transduction mechanism.     

Characterization of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat brain

Rat brain homogenates contain significant amounts of inositol 1,4,5-trisphosphate phosphatase in both 180,000xg (60 min) particulate and supernatant fractions. As other membrane-bound enzymes (e.g. guanylate cyclase), particulate inositol 1,4,5-trisphosphate phosphatase activity is highly sensitive to low concentrations of Triton X-100 (0.03%). Higher concentrations of detergent (1%) partially solubilized the enzyme. Thiol blocking agents (e.g. p-hydroxymercuribenzoate) inactivate inositol 1,4,5-trisphosphate phosphatase activity (an effect reversed with 2-mercaptoethanol). It is thus suggested that enzymatic activity requires the presence of -SH groups.     

Synthetic inositol 1,3,4,5-tetrakisphosphate analogs and their effect on the binding to microsomal fraction of rat cerebellum.

Binding activity of [3H]inositol 1,3,4,5-tetrakisphosphate (InsP4) was characterized with rat cerebellar membranes. Two types of InsP4 analog with either the aminobenzoyl or the aminocyclohexanecarbonyl group on the 2nd position of InsP4 have been synthesized and their effects on the binding activity were also examined. [3H]InsP4 binding was gradually displaced by increasing amounts of unlabeled InsP4, with an IC50 of 60-170 nM, depending on the pH values. The binding was sharply increased at acidic pH and millimolar concentrations of Ca2+, this being in clear contrast with [3H]InsP3 binding noted in the same species of tissue. Heparin inhibited the binding, with an IC50 of 1.7, 3 or 20 micrograms/ml at pH 8.3, 7.2 or 5.0, respectively. Adenine nucleotide inhibited the binding more potently than did [3H]InsP3 binding. InsP4 analogs were as effective as InsP4 in displacing [3H]InsP4 from rat cerebellar membranes, thereby indicating that the 2nd hydroxyl group may not be involved in recognition of InsP4 by its binding sites.     

Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate inhibit inositol-1,3,4,5-tetrakisphosphate 3-phosphatase in rat parotid glands

In assays containing a physiological concentration of inositol 1,3,4,5-tetrakisphosphate (1 microM), this isomer was attacked by both 3- and 5-phosphatases present in rat parotid homogenates and 100,000 X g supernatant and particulate fractions. As the concentration of cytosolic protein in the assay was decreased, the specific activity of the soluble 3-phosphatase increased significantly. In contrast, the specific activity of particulate 3-phosphatase was independent of protein concentration. At the lowest protein concentrations tested, the sum of soluble and particulate 3-phosphatase specific activities was 2.5-fold greater than that of the parent homogenate. These observations indicate that parotid cytosol contains a hitherto undescribed endogenous mechanism for inhibiting 3-phosphatase. The effects upon 3- and 5-phosphatase of a number of inositol polyphosphates were studied. Both activities were inhibited by inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate (IC50 approximately 50 microM). Inositol 3,4,5,6-tetrakisphosphate was a more potent inhibitor of 3-phosphatase (IC50 about 10 microM) and did not affect 5-phosphatase. Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate were very potent inhibitors of 3-phosphatase (IC50 values of 1 and 0.5 microM, respectively); these polyphosphates did not affect 5-phosphatase activity at concentrations of up to 10 microM. Inositol 1,3,4,5,6-pentakisphosphate was a competitive inhibitor of the 3-phosphatase, whereas inositol hexakisphosphate was a mixed inhibitor. These data lead to the proposal that the inositol 1,3,4,5-tetrakisphosphate 3-phosphatase is unlikely to be an important enzyme activity in vivo.     

Inositol 1,3,4,5-tetrakisphosphate stimulates the initiation of DNA synthesis in Ca2+-deprived rat liver cells

The G1-S boundary of non-neoplastic cells requires extracellular Ca2+ for successful transition. Inositol 1,3,4,5-tetrakisphosphate but not inositol 1,4,5-trisphosphate can partially replace Ca2+ and stimulate the initiation of DNA synthesis of Ca2+-deprived T51B rat liver cells but only if sufficient extracellular Ca2+ (i.e., 0.075 mM) is present. The potent tumor promoter and protein kinase C activator 12-O-tetradecanoylphorbol acetate is also capable of replacing extracellular Ca2+ and partially stimulating the initiation of DNA synthesis. In addition, both inositol-1,3,4,5-tetrakisphosphate and 12-O-tetradecanoylphorbol acetate added together elicit a full DNA synthetic response.     

Purification of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain

The ATP-dependent, calmodulin-sensitive 3-kinase responsible for the conversion of D-myo-inositol 1,4,5-trisphosphate to D-myo-inositol 1,3,4,5-tetrakisphosphate has been purified 2,700-fold from rat brain to a specific activity of 2.3 mumol/min/mg protein. A method of purification is described involving chromatography on phosphocellulose, Orange A dye ligand, calmodulin agarose, and hydroxylapatite columns. Neither the highly purified enzyme nor enzyme eluting from the phosphocellulose column were activated by Ca2+. However, enzyme in the 100,000 x g supernatant from rat brain was activated by Ca2+ over the range from 10(-7) to 10(-6) M and Ca2+ sensitivity of the purified enzyme was restored by the addition of calmodulin. The enzyme has a catalytic subunit Mr of 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size exclusion chromatography of the purified enzyme on a Superose 12 column gave a Mr value of 70,000, indicating that the purified enzyme was present as a monomer. In contrast, the 100,000 x g supernatant and the purified enzyme after addition of calmodulin and 10(-6) M Ca2+ chromatographed on size exclusion chromatography with a Mr of 150,000-160,000. These results imply that the native enzyme is a dimeric structure of two catalytic subunits plus calmodulin. The purified enzyme showed a Km of 0.21 +/- 0.08 microM for D-myo-inositol 1,4,5-trisphosphate and had a pH optimum of 8.5. Addition of calmodulin increased both the Km and the Vmax of the purified enzyme about 2-fold. The high affinity of the 3-kinase for D-myo-inositol 1,4,5-trisphosphate together with its activation by Ca2+/calmodulin suggests that this enzyme may exert an important regulatory role in inositol phosphate signaling by promoting the formation of additional inositol polyphosphate isomers.     

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices: Differential effects ofmyo-inositol

To investigate the effects of increasing concentrations ofmyo-inositol (inositol) on receptor stimulated [³H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [³H]inositol (1 to 30 M). Carbachol stimulated formation of [³H]inositol trisphosphate (InsP₃) and [³H]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P₄} increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 M inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 M), [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 M inositol, carbachol stimulated [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 M inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [³H]Ins(1,3,4,5)P₄ formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P₄ mass formation by seven fold. Furthermore, Li⁺ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P₄ mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P₄. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li⁺, increasing the inositol concentration greatly increases the stimulated component of [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P₄ formation is not reflected as an increase in mass formation.     

Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4, 5-trisphosphate-receptor protein from brain, is induced by inositol 1,3,4,5-tetrakisphosphate and regulated by a membrane-associated 5-phosphatase.

The highly conserved 42-kDa protein, p42IP4 was identified recently from porcine brain. It has also been identified similarly in bovine, rat and human brain as a protein with two pleckstrin homology domains that binds Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 with high affinity and selectivity. The brain-specific p42IP4 occurs both as membrane-associated and cytosolic protein. Here, we investigate whether p42IP4 can be translocated from membranes by ligand interaction. p42IP4 is released from cerebellar membranes by incubation with Ins(1,3,4,5)P4. This dissociation is concentration-dependent (> 100 nM), occurs within a few minutes and and is ligand-specific. p42IP4 specifically associates with PtdIns(3, 4,5)P3-containing lipid vesicles and can dissociate from these vesicles by addition of Ins(1,3,4,5)P4. p42IP4 is only transiently translocated from the membranes as Ins(1,3,4,5)P4 can be degraded by a membrane-associated 5-phosphatase to Ins(1,3,4)P3. Then, p42IP4 re-binds to the membranes from which it can be re-released by re-addition of Ins(1,3,4,5)P4. Thus, Ins(1,3,4,5)P4 specifically induces the dissociation from membranes of a PtdIns(3,4,5)P3 binding protein that can reversibly re-associate with the membranes. Quantitative analysis of the inositol phosphates in rat brain tissue revealed a concentration of Ins(1,3,4,5)P4 comparable to that required for p42IP4 translocation. Thus, in vivo p42IP4 might interact with membranes in a ligand-controlled manner and be involved in physiological processes induced by the two second messengers Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3.     

Nuclear magnetic resonance spectroscopic analysis of myo-inositol phosphates including inositol 1,3,4,5-tetrakisphosphate

1H and 31P NMR spectra of a variety of phosphorylated myo-inositols have been analyzed using a Bruker WH-360 spectrometer. Proton and phosphorus chemical shifts and coupling constants are reported for myo-inositol 1-phosphate, myo-inositol 2-phosphate, myo-inositol 5-phosphate, myo-inositol 1,2-cyclic phosphate, myo-inositol 1,4-bisphosphate, myo-inositol 1,4,5-trisphosphate, and myo-inositol 1,3,4,5-tetrakisphosphate. These data provide the basis for the chemical identification and characterization of biologically relevant inositol phosphates.     

Purification of an inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity from rat liver and the evaluation of its substrate specificity.

Hepatic inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity was detected in a 100,000 x g soluble fraction and a detergent-solubilized particulate fraction. Activity in both fractions increased up to 40-fold after anion-exchange chromatography due to removal of endogenous inhibitors (Hodgson, M.E., and Shears, S.B. (1990) Biochem. J. 267, 831-834); at this stage the detergent-solubilized particulate activity comprised over 90% of total activity. The particulate phosphatase was further purified by affinity chromatography using heparin-agarose and red-agarose. The latter column resolved two peaks of enzyme activity (designated 1 and 2 by their order of elution from the column). Their proportions varied between experiments, but peak 2 generally predominated and so this was further purified by hydroxylapatite chromatography. The final preparation was typically 38,000-fold purified with a 7% yield. The apparent molecular mass of this enzyme was 66 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The enzyme had little or no affinity for the following: inositol (1,3,4,6)-tetrakisphosphate, inositol (1,3,4)-trisphosphate, inositol (1,3)-bisphosphate, inositol (3,4)-bisphosphate, and para-nitrophenylphosphate. At pH 7.4 the Km for inositol (1,3,4,5)-tetrakisphosphate was 130 nM and the Vmax was 4250 nmol/mg protein/min. The purified enzyme also dephosphorylated inositol (1,3,4,5,6)-pentakisphosphate to inositol (1,4,5,6)-tetrakisphosphate (Km = 40 nM, Vmax = 211 nmol/mg protein/min), and inositol hexakisphosphate to at least five isomers of inositol pentakisphosphate (Km = 0.3 nM, Vmax = 12 nmol/mg protein/min). The latter affinity is the highest yet defined for an enzyme involved in inositol phosphate metabolism. Determinations of IC50 values, and Dixon plots, revealed that with the (1,3,4,5)-tetrakisphosphate as substrate, the pentakis- and hexakisphosphates were potent competitive inhibitors; the Ki values (25 and 0.5 nM, respectively) were similar to their substrate Km values. The kinetic properties of this enzyme, as well as estimates of the cellular levels of its potential substrates, indicate that inositol pentakisphosphate and inositol hexakisphosphate are likely to be the preferred substrates in vivo.