Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets.
The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.
D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000097:blood*; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000097:blood*; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000097:blood* / Q000302:isolation & purification
D007527:Isoenzymes; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D002154:Calpain; D002118:Calcium