Biochemical and Pharmacologic Characterization of a Phosphatidylinositol-Specific Phospholipase C in Rat Neutrophils

The phosphatidylinositol (P1)-specific phospholipase C (PLC) activity contained in soni- cates of casein-elicited (4-6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (P1) as the substrate, PLC activity is found both in the super- nate and pellet of a 100,000g spin of the neutrophil sonicate. Further fractionation of the crude sonicate by centrifugation on discontinuous sucrose gradients indicates that the PLC activity is predominantly cytosolic with lesser amounts of activity found in the plasma membrane and granule enriched fractions. Hydrolysis of P1 by the sonicate PLC is linear for 15-20 mm at 37#{176}C and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for P1with little, if any, hydrolytic activity towards other phospholipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylcholine (PC). The PLC activity has a pH optimum of 5.5-6.0, is enhanced 1.5-3-fold by the addition of deoxycholate, and is Ca + + dependent. Kinetic analysis of the PLC hydrolysis of P1 yields an apparent Km of 240 ± 85 /LM and a Vmax of 34.3 ± 17.0 nmol/min/mg protein (n = 3). Similarly, when phosphatidylinositol 4,5 bisphosphate (PIP2) is used as substrate, an apparent Km of 109 ± 66 /2M and a Vmax of 14.3 ± 10.4 nmol/min/mg (n = 3) protein is obtained. These data suggest that PIP2 may be a slightly better substrate for the PMN PLC relative to P1. Finally, a variety of drugs previously reported to inhibit platelet PLC activity in vitro were tested for their ability to inhibit rat PMN PLC. Of the compounds tested, none were potent (i.e., IC50 values � 100 /LM) inhibitors of the PMN PLC.