Identification and characterization of the phosphatidylinositol-(4,5)- bisphosphate 5-phosphatase in human platelets

Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)-P₂) is the precursor of several second messenger molecules. In unstimulated cells PtdIns(4,5)P₂ is hydrolyzed by a PtdIns(4,5)P₂ 5-phosphatase to form phosphatidylinositol 4-phosphate (PtdIns(4)P) which is subsequently recycled to phosphatidylinositol. PtdIns(4,5)P₂ 5-phosphatase activity was detected in platelet cytosolic and particulate fractions. The platelet PtdIns(4,5)P₂5- phosphatase activity was magnesium but not calcium dependent. The elution profile of platelet cytosolic PtdIns(4,5)P₂ 5-phosphatase from anion exchange resins, exactly matched that of the 75-kDa inositol-polyphosphate 5- phosphatase (Ins(1,4,5)P₃ 5-phosphatase). The latter is a signal terminating enzyme responsible for the hydrolysis of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P₃) to inositol (1,4)-bisphosphate (Mitchell, C. A., Connolly, T. M., and Majerus, P. W. (1989) J. Biol. Chem. 264, 8873-8877). Polyclonal antibodies raised against recombinant 75-kDa Ins(1,4,5)P₃ 5-phosphatase specifically immunoprecipitated all PtdIns(4,5)P₂ 5-phosphatase activity from both the platelet membrane and cytosolic fractions. Purified 75-kDa Ins(1,4,5)P₃ 5-phosphatase hydrolyzed PtdIns(4,5)P₂ forming PtdIns(4)P (K(m) = 250 μM). By contrast, purified membrane-associated 43-kDa Ins(1,4,5)P₃ 5-phosphatase did not hydrolyze PtdIns(4,5)P₂. In the unstimulated platelet, recycling of PtdIns(4,5)P₂ to PtdIns(4)P is mediated by the 75-kDa Ins(1,4,5)P₃ 5-phosphatase.