Identification of polyphosphoinositide-specific phospholipase C and its resolution from phosphoinositide-specific phospholipase C from human platelet extract.
Sonicated platelet extract, obtained after high-speed centrifugation, was fractionated by phosphocellulose chromatography, and assayed for phosphodiesteric cleavage of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP2) using (i) single bilayer vesicles from total platelet membrane lipids and (ii) single bilayer vesicles from individual pure lipids. When total platelet lipids were used as the substrate, two clear-cut, but incompletely resolved, peaks of phosphoinositide-specific phospholipase C (PI-PLC) were detected; one of these activities hydrolysed PIP alone, whereas the second peak hydrolysed all three phosphoinositides. When pure phosphoinositides were used as the substrate, three distinct peaks of PI-PLC were observed; two of these peak activities correspond to the peaks when assays were conducted with total platelet membrane lipids. The third peak of activity hydrolysed PIP2 and PIP, but not PI. These results clearly show that there exist PIP/PIP2-specific PLC enzymes that do not act on PI, and escaped detection thus far because of the general practice of using PI as the substrate to monitor PI-PLC activities. I propose that these PIP/PIP2-specific PLC enzymes are the real candidates that respond to agonist occupation of receptors on the cell surface to generate the two putative second messengers inositol triphosphate and diacylglycerol.
Manne
V
V
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
D001792:Blood Platelets / Q000201:enzymology*; D002845:Chromatography / Q000379:methods; D006801:Humans; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000097:blood* / Q000378:metabolism
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010738:Type C Phospholipases