Purification and properties of a human platelet inositol 1,4,5-trisphosphate 3-kinase.
An inositol 1,4,5-trisphosphate 3-kinase (Ins(1,4,5)P3 3-kinase) has been purified 943-fold from a 30,000g human platelet extract and has a specific activity of 283 nmol/min/mg protein and an apparent Km for inositol 1,4,5-trisphosphate of 0.76 microM; the optimal pH for the enzymatic activity was 7.2. Under both denaturing and nondenaturing conditions, the kinase preparation contained two polypeptides, both of which exhibited Ca2+/calmodulin-dependent Ins(1,4,5)P3 3-kinase activity. In the presence and absence of calmodulin, Ins(1,4,5)P3 3-kinase exhibited a biphasic response to Ca2+, being stimulated between 10(-7) and 10(-6) M Ca2+ and inhibited when the Ca2+ level was further increased. Ins(1,4,5)P3 3-kinase was stimulated by calmodulin approximately 10-fold, requiring 55 nM calmodulin for a half-maximal effect. Calmodulin stimulation was immediately reversed upon chelation of Ca2+ by ethylene glycol bis (beta-amino-ethyl ether) N,N'-tetraacetic acid consistent with a mechanism of activation involving a direct interaction of calmodulin with Ins(1,4,5)P3 3-kinase. Since we have previously shown that Ins(1,4,5)P3 3-kinase can also be phosphorylated and consequently inactivated by protein kinase C in vitro (Lin, A. N., Barnes, S., and Wallace, R. W., 1990, Biochem. Biophys. Res. Commun. 170, 1369-1376), Ins(1,4,5)P3 3-kinase appears to be a key enzyme in the inositol phosphate signaling pathway and as such may play an important role in human platelet function.
Department of Pharmacology, University of Alabama, Birmingham 35294.
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Lin
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Wallace
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Barnes
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D010770:Phosphotransferases / Q000097:blood* / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor); D011092:Polyethylene Glycols; D011489:Protein Denaturation; D013552:Swine
D002147:Calmodulin; D011092:Polyethylene Glycols; D004533:Egtazic Acid; D017830:Octoxynol; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium