Characterization of partially purified phospholipase C from human platelet membranes.
A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.
Banno
Y
Y
Department of Biochemistry, Gifu University School of Medicine, Japan.
Nozawa
Y
Y
D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D003840:Deoxycholic Acid; D006801:Humans; D066298:In Vitro Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000097:blood; D010738:Type C Phospholipases / Q000097:blood* / Q000302:isolation & purification
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D003840:Deoxycholic Acid; D010738:Type C Phospholipases; D002118:Calcium