Purification and properties of a human platelet inositol 1,4,5-trisphosphate 3-kinase.

Academic Article

Abstract

  • 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.
  • Keywords

  • Animals, Blood Platelets, Calcium, Calmodulin, Chromatography, Chromatography, High Pressure Liquid, Egtazic Acid, Electrophoresis, Polyacrylamide Gel, Humans, Hydrogen-Ion Concentration, Kinetics, Molecular Weight, Octoxynol, Phosphotransferases, Phosphotransferases (Alcohol Group Acceptor), Polyethylene Glycols, Protein Denaturation, Swine
  • Digital Object Identifier (doi)

    Author List

  • Lin A; Wallace RW; Barnes S
  • Start Page

  • 412
  • End Page

  • 420
  • Volume

  • 303
  • Issue

  • 2