Phosphatidylinositol-specific phospholipase C of murine lymphocytes.
Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. The activity of the cytosolic enzyme was first detected at 1 microM Ca2+, and maximum activity was observed at 100 microM Ca2+, while the membrane-bound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate.A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an alpha-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D005260:Female; D008214:Lymphocytes / Q000201:enzymology*; D051379:Mice; D008813:Mice, Inbred ICR; D010716:Phosphatidylinositols / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*
D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D002118:Calcium