Partial purification and characterization of membrane-bound and cytosolic phosphatidylinositol-specific phospholipases C from murine thymocytes.
Membrane-bound and cytosolic phosphatidylinositol (PI)-specific phospholipases C in murine thymocytes have been partially purified and characterized. The membrane-bound enzyme was extracted from microsomes with sodium cholate and purified by sequential column chromatographies on Sephadex G-100, heparin-Sepharose CL-6B, and Sephadex G-100. The cytosolic enzyme was purified from the cytosol by sequential column chromatographies on Sephadex G-100 and FPLC-Mono S. Specific activities of the membrane-bound enzyme and the cytosolic enzyme increased more than 1,800- and 1,400-fold, respectively, compared with those of microsomes and the cytosol. The molecular weights of the both enzymes were estimated to be about 70,000 by gel filtration. These purified enzymes also hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2). At neutral pH and low Ca2+ concentrations, the membrane-bound enzyme hydrolyzed PIP2 in preference to PI and showed higher activity than the cytosolic enzyme. These activities were also affected differently by various lipids. For PIP2 hydrolysis, all lipids investigated except lysophosphatidylcholine enhanced the activity of the membrane-bound enzyme, while phosphatidylcholine (PC) and phosphatidylserine (PS) did not significantly affect the activity of the cytosolic enzyme. PC, PE, and PS inhibited the activities of the membrane-bound and cytosolic enzymes for PI hydrolysis. The physiological implications of these results are discussed.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D010716:Phosphatidylinositols / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology*; D013950:Thymus Gland / Q000201:enzymology*; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism
D010716:Phosphatidylinositols; D010743:Phospholipids; D010738:Type C Phospholipases