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)P2 5-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 (Km = 250 microM). 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.
Matzaris
M
M
Department of Medicine, Monash Medical School, Box Hill Hospital, Melbourne, Australia.
Jackson
S P
SP
Laxminarayan
K M
KM
Speed
C J
CJ
Mitchell
C A
CA
D001792:Blood Platelets / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D003599:Cytoskeleton / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000097:blood / Q000302:isolation & purification; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000097:blood* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis; D013347:Subcellular Fractions / Q000201:enzymology; D000099182:Phosphoinositide 5-Phosphatases
D007527:Isoenzymes; D010744:Phosphoric Monoester Hydrolases; D011994:Recombinant Proteins; D000099182:Phosphoinositide 5-Phosphatases