Kinetic consequences of the inhibition by ATP of the metabolism of inositol (1,4,5) trisphosphate and inositol (1,3,4,5) tetrakisphosphate in liver. Different effects upon the 3- and 5-phosphatases

A kinetic analysis was undertaken of the inhibition by 5 mM MgATP of Ins(1,4,5)P3 5-phosphatase in 100,000 g particulate fractions prepared from liver homogenates. The Km for Ins(1,4,5)P3 was increased by 44% (from 16 to 23 microM). The competitive nature of the inhibition was confirmed with a Dixon plot. The effect of MgATP on 5-phosphatase was also studied at physiological concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (i.e. 1.5 microM); the rate of substrate hydrolysis was inhibited by over 30%. Ins(1,3,4,5)P4 was also hydrolysed by a 3-phosphatase, but this enzyme was unaffected by 5 mM MgATP. Thus, ATP, by differentially affecting Ins(1,3,4,5)P4 3- and 5-phosphatase, may increase the flux through the futile cycle that interconverts Ins(1,4,5)P3 and Ins(1,3,4,5)P4.     

The isolation and characterization of inositol polyphosphate 4-phosphatase

We previously identified an alternative pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) in calf brain. The enzyme responsible for the degradation of Ins(1,3,4)P3 was designated as inositol polyphosphate 4-phosphatase (Bansal, V. S., Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 9644-9647). We have now purified this enzyme 3390-fold from calf brain-soluble fraction. The isolated enzyme has an apparent molecular mass of 110 kDa as determined by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme migrates as a protein of 105 kDa, suggesting that it is monomeric. Among various 4-phosphate-containing inositol polyphosphates, the enzyme hydrolyzes only Ins(1,3,4)P3 and inositol 3,4-bisphosphate (Ins(3,4)P2), yielding inositol 1,3-bisphosphate and inositol 3-phosphate as products. The inositol polyphosphate 4-phosphatase has apparent Km values of 40 and 25 microM for Ins(1,3,4)P3 and Ins(3,4)P2, respectively. The maximum velocities for these two substrates are 15-20 mumol of product/min/mg protein. Ins(1,3,4)P3 is a competitive inhibitor of Ins(3,4)P2 hydrolysis with an apparent Ki of 27 microM implying that the same active site is involved in hydrolysis of both substrates. The final enzyme preparation retained a small inositol polyphosphate 3-phosphatase activity (less than 2% of rate of inositol polyphosphate 4-phosphatase activity) which most likely reflects a contaminant. The enzyme displays maximum activity between pH 6.5 and 7.5. It is not inhibited by Li+, Ca2+, or Mg2+ except at 10 mM divalent ions. Mn2+ inhibits enzyme at high concentrations IC50 = 1.5 mM.     

Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.     

Hydrolysis of inositol phosphates by plant cell extracts.

A gel-filtered soluble fraction prepared from suspension-cultured Nicotiana tabacum cells hydrolysed inositol mono-, bis- and tris-phosphates. At a concentration of 7.5 microM the rates of hydrolysis followed the sequence Ins(1,4,5)P3 greater than Ins(1,4)P2 greater than Ins(4)P congruent to Ins(1)P. The major products of Ins(1,4,5)P3 hydrolysis identified by h.p.l.c. were Ins(1,4)P2 and Ins(4,5)P2. Ins(1,4)P2 was hydrolysed exclusively to Ins(4)P. The inclusion of Ca2+ in the incubation buffer markedly stimulated the hydrolysis of all the inositol phosphate substrates. Under identical conditions, Ca2+ inhibited the hydrolysis of inositol phosphates by soluble extracts prepared from rat brain. Half-maximal stimulation of Ins(1,4)P2 hydrolysis was obtained at free [Ca2+] of 0.6 and 1.2 microM when the Mg2+ concentration in the incubations was 0.3 and 1.0 mM respectively. This effect of Ca2+ was exerted solely by increasing the Vmax. of hydrolysis without affecting the Km for Ins(1,4)P2. Again, in contrast with brain, the hydrolysis of inositol bis- or mono-phosphates was insensitive to high concentrations of Li+. We conclude that plants contain specific Li+-insensitive inositol phosphate phosphatases that are regulated by low concentrations of Ca2+ in a manner which is different from that observed in mammalian tissues.     

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.     

Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells.

Dictyostelium discoideum homogenates contain phosphatase activity which rapidly dephosphorylates Ins(1,4,5)P3 (D-myo-inositol 1,4,5-trisphosphate) to Ins (myo-inositol). When assayed in Mg2+, Ins(1,4,5)P3 is dephosphorylated by the soluble Dictyostelium cell fraction to 20% Ins(1,4)P2 (D-myo-inositol 1,4-bisphosphate) and 80% Ins(4,5)P2 (D-myo-inositol 4,5-bisphosphate). In the particulate fraction Ins(1,4,5)P3 5-phosphatase is relatively more active than the Ins(1,4,5)P3 1-phosphatase. CaCl2 can replace MgCl2 only for the Ins(1,4,5)P3 5-phosphatase activity. Ins(1,4)P2 and Ins(4,5)P2 are both further dephosphorylated to Ins4P (D-myo-inositol 4-monophosphate), and ultimately to Ins. Li+ ions inhibit Ins(1,4,5)P3 1-phosphatase, Ins(1,4)P2 1-phosphatase, Ins4P phosphatase and L-Ins1P (L-myo-inositol 1-monophosphate) phosphatase activities; Ins(1,4,5)P3 1-phosphatase is 10-fold more sensitive to Li+ (half-maximal inhibition at about 0.25 mM) than are the other phosphatases (half-maximal inhibition at about 2.5 mM). Ins(1,4,5)P3 5-phosphatase activity is potently inhibited by 2,3-bisphosphoglycerate (half-maximal inhibition at 3 microM). Furthermore, 2,3-bisphosphoglycerate also inhibits dephosphorylation of Ins(4,5)P2. These characteristics point to a number of similarities between Dictyostelium phospho-inositol phosphatases and those from higher organisms. The presence of an hitherto undescribed Ins(1,4,5)P3 1-phosphatase, however, causes the formation of a different inositol bisphosphatase isomer [Ins(4,5)P2] from that found in higher organisms [Ins(1,4)P2]. The high sensitivity of some of these phosphatases for Li+ suggests that they may be the targets for Li+ during the alteration of cell pattern by Li+ in Dictyostelium.     

Inositol-1,2-cyclic-phosphate 2-inositolphosphohydrolase. Substrate specificity and regulation of activity by phospholipids, metal ion chelators, and inositol 2-phosphate.

Glycerophosphoinositol (GroPIns) is a major inositol phosphate in many cell types. In this study we have determined the optimal conditions (pH 8.0 and 0.5 mM MnCl2) for the metabolism of this molecule in an extract from human placenta, and we show that the major product is inositol (1)-phosphate (Ins(1)P). The enzyme activity that catalyzes this reaction is contained in the same protein designated previously as inositol-(1,2)-cyclic-phosphate 2-inositolphosphohydrolase (cyclic hydrolase), a phosphodiesterase that catalyzes the conversion of inositol-(1,2)-cyclic phosphate (cIns(1,2)P) to Ins(1)P. In addition, the enzyme also catalyzes the production of Ins(1)P from inositol (1)-methylphosphate. All of these substrates, (cIns(1,2)P, GroPIns, and inositol (1)-methylphosphate), contain a phosphodiester bond at the 1-position of the inositol ring. Additional phosphate groups on the 4- or 5-positions of the inositol ring prevent hydrolysis by cyclic hydrolase. The Km of the enzyme for GroPIns is 0.67 mM, and the Vm is 5 mumol/min/mg of protein. GroPIns competitively inhibits cIns(1,2)P hydrolysis with a Ki equal to its Km as a substrate. Hydrolysis of GroPIns and cIns(1,2)P is stimulated by MnCl2, phosphatidylserine, and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). However, whereas cIns(1,2)P hydrolysis is increased 5-8-fold by phosphatidylserine and EGTA only a 2-fold increase of GroPIns hydrolysis occurs under the same conditions. Hydrolysis of both GroPIns and cIns(1,2)P is inhibited by Ins(2)P; the ID50 values are 12 and 1 microM, respectively. There are significant quantities of GroPIns and Ins(2)P in 3T3 cells, indicating that these compounds that alter cIns(1,2)P hydrolase activity may modulate intracellular levels of cIns(1,2)P. Finally, we present evidence suggesting that the substrate specificity of this enzyme is altered during cell transformation.     

Kinetic and inhibitor profiles of soluble and particulate inositol 1,4-5-trisphosphate 5-phosphatase from GH3 and IMR-32 cells.

A simple procedure for assay of Ins(1,4,5)P3 5-phosphatase is described. The reaction products [( 3H]Ins(1,4)P2, [3H]InsP and myo-[3H]inositol) are completely separated from one another, with quantitative yield, on Amprep SAX (100 mg) minicolumns. [3H]Ins(1,4,5)P3 [and [3H]Ins(1,3,4,5)P4] are adsorbed to the columns but not released to any appreciable extent by the elution conditions used. In GH3 cells, the stepwise dephosphorylation of [3H]Ins(1,4,5)P3 to myo-[3H]inositol was demonstrated, and was inhibited by 2.3-bisphosphoglycerate. The Km of the soluble form of the enzyme was lower in GH3 cells (8-13 microM) than in IMR-32 cells (26-32 microM) or in rat cerebral-cortical samples (22 microM. The Km of the particulate form of the enzyme was similar in all three preparations (10-16 microM). The pH profiles of the two soluble 5-phosphatases differed, with a wider pH optimum for the GH3-cell activity than for the IMR-32-cell activity. The soluble and particulate GH3 enzymes were more sensitive than the corresponding IMR-32 enzymes to inhibition by p-hydroxymercuribenzoate, whereas there were no differences in their sensitivities to glucose 6-phosphate, 2,3-bisphosphoglycerate, fructose 1.6- and 2.6-bisphosphate and non-radioactive Ins(1,3,4,5)P4. Dialysis of the soluble fractions and washing of the particulate fractions did not affect the inhibitor sensitivities, except for the soluble IMR-32 fraction and p-hydroxymercuribenzoate. The Km value of the soluble GH3 5-phosphatase activity was lower, and the inhibition by Ins(1,3,4,5)P4 greater, after adsorption to and elution from phosphocellulose. It is concluded that there are qualitative differences in the properties of the soluble 5-phosphatase activity from GH3 and IMR-32 cells.     

The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase

Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) metabolism has been studied in liver homogenates and in 100,000 x g supernatant and particulate fractions. When liver homogenates were incubated in an "intracellular" medium containing 5 mM MgATP, equal proportions of Ins(1,3,4)P3 were dephosphorylated and phosphorylated. Two inositol tetrakisphosphate (InsP4) products and an inositol pentakisphosphate (InsP5) were detected. The InsP4 isomers were unequivocally identified as inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by high performance liquid chromatography separation of inositol phosphates, periodate oxidation, alkaline hydrolysis, and stereo-specific polyol dehydrogenase. Ins(1,3,4)P3 5-kinase is a novel enzyme activity and accounted for 16% of the total Ins(1,3,4)P3 phosphorylation. Ins(1,3,4,6)P4 was also shown to be further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) by a kinase not previously known to occur in liver. About 75% of Ins(1,3,4)P3 kinase activities were soluble and were partly purified by anion-exchange fast protein liquid chromatography. The two Ins(1,3,4)P3 kinase activities eluted as a single peak that was well resolved from Ins(1,3,4)P3 phosphatase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5)P4 5-phosphatase activities. A further novel observation was that 10 microM Ins(1,3,4,5)P4 inhibited Ins(1,3,4)P3 kinase activities by 60%.     

Properties of inositol polyphosphate 1-phosphatase

We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 15946-15952). We have now purified the enzyme to homogeneity from calf brain. The enzyme hydrolyzes 50.3 mumol of Ins(1,4)P2/min/mg protein. The enzyme has an apparent mass of 44,000 daltons as determined both by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that it is monomeric. Lithium ions inhibit Ins(1,3,4)P3 hydrolysis uncompetitively with an apparent Ki of approximately 0.3 mM LiCl. Calcium inhibits hydrolysis of Ins(1,4)P2 and Ins(1,3,4)P3 equally, with approximately 40% inhibition occurring at 1 microM free Ca2+. Rabbit polyclonal antiserum against purified inositol polyphosphate 1-phosphatase was prepared which immunoprecipitates approximately 0.3 milliunits of activity/microliter serum (1 unit = 1 mumol of Ins(1,4)P2 hydrolyzed per min). This antiserum was used to determine the enzyme content in several bovine tissues, all of which had a similar intrinsic specific activity (i.e. approximately 0.3 milliunits/microliter antiserum). Tissues studied included brain, heart, kidney, liver, lung, parotid, spleen, testis, and thymus. Approximately 10-15% of the total inositol polyphosphate 1-phosphatase activity in calf brain homogenates remains in a particulate fraction; antiserum also binds 0.3 milliunits of membrane-associated activity/microliter antiserum. Thus, a single enzyme can account for Ins(1,4)P2 hydrolytic activity in the bovine tissues. Ins(1,3,4)P3 metabolism was also investigated in bovine tissue homogenates. Inositol polyphosphate 1-phosphatase accounts for greater than 80% of the hydrolytic activity in all tissues studied except brain, where inositol polyphosphate 4-phosphatase is the major enzyme that hydrolyzes Ins(1,3,4)P3. The apparent Km of inositol polyphosphate 1-phosphatase for Ins(1,3,4)P3 varies approximately 3-4-fold among the bovine tissues.     

The metabolism of inositol 1,3,4-trisphosphate to inositol 1,3-bisphosphate

We previously demonstrated a pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) to inositol 3,4-bisphosphate (Ins(3,4)P2) in calf brain extracts. Inositol polyphosphate 1-phosphatase, a Mg2+-dependent, lithium ion-inhibited enzyme, specifically hydrolyzes Ins(1,3,4)P3 to Ins(3,4)P2 and Ins(1,4)P2 to Ins 4-P (Inhorn, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). Now we have found an alternative pathway for the metabolism of Ins(1,3,4)P3 in crude calf brain extracts. Along this pathway, Ins(1,3,4)P3 is first converted to Ins(1,3)P2 which is further hydrolyzed to Ins 1-P. This pathway involves a 4-phosphatase and a 3-phosphatase which do not require Mg2+ and are not inhibited by lithium ions. A similar 4-phosphatase also degrades Ins(3,4)P2 to Ins 3-P. Three different inositol bisphosphates formed from calf brain supernatant are each further metabolized by a separate enzyme. The three inositol monophosphates, i.e. Ins 1-P, Ins 3-P, and Ins 4-P, are converted to inositol by inositol monophosphate phosphatase (Ackermann, K. E., Gish, B. G., Honchar, M. P., and Sherman, W. R. (1987) Biochem. J. 242, 517-524).     

Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase in rat and bovine brain tissues

Inositol 1,4,5-trisphosphate (Ins P3) 3-kinase catalyzes the ATP-dependent phosphorylation of Ins P3 to Inositol 1,3,4,5-tetrakisphosphate (Ins P4). Ca2+/calmodulin (CaM)-sensitivity of Ins P3 3-kinase was measured in the crude soluble fraction from rat brain and different anatomic regions of bovine brain. Kinase activity was inhibited in the presence of EGTA (free Ca2+ below 1 nM) as compared to Ca2+ (10 microM free Ca2+) or Ca2+ (10 microM free Ca2+) and CaM (1 microM). Ca2+-sensitivity was also seen for the cAMP phosphodiesterase measured under the same assay conditions, but was not for the Ins P3 5-phosphatase. DEAE-cellulose chromatography of the soluble fraction of rat brain or bovine cerebellum resolved a Ca2+/CaM-sensitive Ins P3 3-kinase (maximal stimulation at 1 microM Ins P3 substrate level was 2.0-3.0 fold).     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

Dephosphorylation of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,3,4-triphosphate.

We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.     

Purification and properties of inositol-1,4-bisphosphatase from bovine brain.

Inositol-1,4-bisphosphatase has been purified 13,000-fold from bovine brain supernatant. The enzyme is monomeric, with an apparent subunit Mr of 40,000. Maximal hydrolytic rates were observed in Tris buffer, pH 7.8, in the presence of 9 mM-Mg2+. The enzyme acted as a 1-phosphatase, hydrolysing both inositol 1,4-bisphosphate [Ins(1,4)P2] (Km 0.04 mM) and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] (Km 0.5 mM) to inositol 4-phosphate and inositol 3,4-bisphosphate respectively. Li+ inhibited the hydrolysis of both substrates in an uncompetitive manner, with apparent Ki values of 9.63 mM and 0.46 mM for Ins(1,4)P2 and Ins(1,3,4)P3 respectively.     

Purification and characterization of two types of soluble inositol phosphate 5-phosphomonoesterases from rat brain

Two soluble forms of inositol phosphate 5-phosphomonoesterase have been partially purified and characterized from rat brain and are referred to as type 1 and type 2 according to their order of elution from DEAE-Sepharose. Together, these enzymes represent 26 +/- 3% (mean +/- S.E., n = 4) of the total inositol 1,4,5-triphosphate (Ins(1,4,5)P3) phosphatase activity assayed in crude brain homogenate and are present in approximately equal total activities in a 100,000 x g supernatant, with the remainder being membrane-bound. Both soluble enzymes require Mg2+ for activity, are moderately inhibited by Ca2+ in the micromolar range, and can be inhibited by millimolar concentrations of a variety of phosphorylated compounds. The type 1 enzyme has been purified to a specific activity of 1.06 mumol/min/mg protein. It elutes as a 60-kDa protein on Sephacryl S-200. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the type 1 enzyme correlates with a pair of protein bands of 66 and 60 kDa. It has apparent Km values of 3 and 0.8 microM for Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), respectively, and hydrolyses Ins(1,4,5)P3 approximately 12 times faster than Ins(1,3,4,5)P4. The type 2 enzyme has been purified to a specific activity of 15.2 mumol/min/mg protein, elutes as a protein of 160 kDa on Sephacryl S-300, and migrates as a similarly sized subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has an apparent Km for Ins(1,4,5)P3 of 18 microM. Its apparent Km for Ins(1,3,4,5)P4, however, is greater than 150 microM, suggesting that this enzyme is primarily an Ins(1,4,5)P3 5-phosphomonoesterase. The relationship of these two enzymes to the inositol tris/tetrakisphosphate pathway is discussed.     

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.     

Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells.

Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.     

Thrombin and phorbol ester stimulate inositol 1,3,4,5-tetrakisphosphate 3-phosphomonoesterase in human platelets

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular Ca2+, is metabolized either by dephosphorylation to inositol 1,4-bisphosphate(Ins-(1,4)P2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). It has been shown in vitro that Ins(1,3,4,5)P4 is also dephosphorylated by a 5-phosphomonoesterase to inositol 1,3,4-trisphosphate. However, we have found that exogenous Ins(1,3,4,5)P4 is dephosphorylated to predominantly Ins(1,4,5)P3 in saponin-permeabilized platelets in the presence of KCl (40-160 mM). This inositol polyphosphate 3-phosphomonoesterase activity is independent of Ca2+ (0.1-100 microM), and it was also observed when the ionic strength of the incubation medium was increased with Na+. The action of KCl appears to be due to activation of a 3-phosphomonoesterase as well as an inhibition of the 5-phosphomonoesterase, because the dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 was completely inhibited by KCl. The 3-phosphomonoesterase may be regulated by a protein kinase C, since both thrombin and phorbol dibutyrate increase 3-phosphomonoesterase activity and this is inhibited by staurosporine. The formation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 reported here provides an additional pathway for the formation of the Ca2+-mobilizing second messenger in stimulated cells.     

Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+

We recently identified an enzyme which we have designated inositol polyphosphate 1-phosphatase that hydrolyzes both inositol 1,3,4-trisphosphate (Ins-1,3,4-P3) and inositol 1,4-bisphosphate (Ins-1,4-P2), yielding inositol 3,4-bisphosphate and inositol 4-phosphate, respectively, as products (Inhorn, R. C., Bansal, V.S., and Majerus, P.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). We have now purified the inositol polyphosphate 1-phosphatase 3600-fold from calf brain supernatant. The purified enzyme has an apparent molecular mass of 44,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphatase activities. The enzyme hydrolyzes Ins-1,4-P2 with an apparent Km of approximately 4-5 microM, while it degrades Ins-1,3,4-P3 with an apparent Km of approximately 20 microM. The enzyme hydrolyzes these substrates at approximately the same maximal velocity. Inositol polyphosphate 1-phosphatase shows a sigmoidal dependence upon magnesium ion, with 0.3 mM Mg2+ causing half-maximal stimulation. A Hill plot of the data is linear with a value of n = 1.9, suggesting that the enzyme binds magnesium cooperatively. Calcium and manganese inhibit enzyme activity, with 50% inhibition at approximately 6 microM. Lithium inhibits Ins-1,4-P2 hydrolysis uncompetitively with a Ki of approximately 6 mM. This mechanism of lithium inhibition is similar to that observed for the inositol monophosphate phosphatase (originally designated myo-inositol-1-phosphatase; Hallcher, L.M., and Sherman, W.R. (1980) J. Biol. Chem. 255, 10896-10901), suggesting that these two enzymes are related. Lithium also inhibits Ins-1,3,4-P3 hydrolysis with an estimated Ki of 0.5-1 mM.