Biochemical and pharmacologic characterization of a phosphatidylinositol-specific phospholipase C in rat neutrophils.
The phosphatidylinositol (PI)-specific phospholipase C (PLC) activity contained in sonicates of casein-elicited (4-6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (PI) as the substrate, PLC activity is found both in the supernate 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 PI by the sonicate PLC is linear for 15-20 min at 37 degrees C and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for PI with 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 PI yields an apparent Km of 240 +/- 85 microM 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 microM 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 PI. 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 less than or equal to 100 microM) inhibitors of the PMN PLC.
Mackin
W M
WM
Medical Products Department, E.I. DuPont de Nemours & Co., Inc., Wilmington, Delaware.
Stevens
T M
TM
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D009504:Neutrophils / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000097:blood*
D010716:Phosphatidylinositols; D003840:Deoxycholic Acid; D010738:Type C Phospholipases; D002118:Calcium