Isolation and characterization of one soluble and two membrane-associated forms of phosphoinositide-specific phospholipase C from human platelets.
Two forms (mPLC-I, mPLC-II) of phosphoinositide-specific phospholipase C have been purified, 1494- and 1635-fold, respectively, from plasma membranes of human platelets. Purified mPLC-I and mPLC-II had estimated molecular weights by gel filtration and sodium dodecyl sulfate-polyacrylamide gels of 69,000 and 63,000, respectively. Two cytosolic forms (PLC-I and PLC-II) of phosphoinositide-specific phospholipase C were also resolved on a phenyl-Sepharose column. The major cytosolic form present in outdated platelets, PLC-II, was purified to homogeneity by chromatography on Fast Q-Sepharose, cellulose phosphate, heparin-agarose, phenyl-Sepharose, Superose 12, DEAE-5PW, and hydroxylapatite. Purified PLC-II had a molecular weight of 57,000 on sodium dodecyl sulfate-polyacrylamide gels. mPLC-I, mPLC-II, and PLC-II hydrolyzed both PI and PIP2. The Vmax for PIP2 hydrolysis was similar for all three forms of PLC and was approximately 5-fold greater than for PI hydrolysis. The Km for PIP2 hydrolysis was also similar for the three enzymes. In contrast, the Km for PI hydrolysis by PLC-II was 10-fold lower than by mPLC-I and mPLC-II. In addition, antibody prepared against PLC-II did not cross-react with either mPLC-I or mPLC-II. These data indicate that platelets contain membrane-associated phosphoinositide-specific phospholipases C that are distinct from at least one cytosolic form (PLC-II) of the enzyme.
Baldassare
J J
JJ
American Red Cross, St. Louis, Missouri 63108.
Henderson
P A
PA
Fisher
G J
GJ
D001792:Blood Platelets / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008566:Membranes / Q000201:enzymology; D008970:Molecular Weight; D010716:Phosphatidylinositols; D012995:Solubility; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000097:blood*
D010716:Phosphatidylinositols; D010738:Type C Phospholipases