Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets.
Two peaks (mPLC-I and mPLC-II) of phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity were resolved when 1% sodium cholate extract from particulate fractions of human platelet was chromatographed on a heparin-Sepharose column. The major peak of enzyme activity (mPLC-II) was purified to homogeneity by a combination of Fast Q-Sepharose, heparin-Sepharose, Ultrogel AcA-44, Mono Q, Superose 6-12 combination column, and Superose 12 column chromatographies. The specific activity increased 2,700-fold as compared with that of the starting particulate fraction. The purified mPLC-II had an estimated molecular weight of 61,000 on sodium dodecyl sulfate-polyacrylamide gels. The minor peak of enzyme activity (mPLC-I) was partially purified to 430-fold. Both enzymes hydrolyzed PIP2 at low Ca2+ concentration (0.1-10 microM) and exhibited higher Vmax for PIP2 than for phosphatidylinositol. PIP2-hydrolyzing activities of both enzymes were enhanced by various detergents and lipids, such as deoxycholate, cholate, phosphatidylethanolamine, and dimyristoylphosphatidylcholine. The mPLC-I and mPLC-II activities were increased by Ca2+, but not by Mg2+, while Hg2+, Fe2+, Cu2+, and La3+ were inhibitory. GTP-binding proteins (Gi, Go, and Ki-ras protein) had no significant effects on the mPLC-II activity.
Banno
Y
Y
Department of Biochemistry, Gifu University School of Medicine, Japan.
Yada
Y
Y
Nozawa
Y
Y
D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology; D009711:Nucleotides / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000097:blood*; D010738:Type C Phospholipases / Q000302:isolation & purification*
D003902:Detergents; D008055:Lipids; D009711:Nucleotides; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium