INHIBITION OF MACROPHAGE CA2+-INDEPENDENT PHOSPHOLIPASE A(2) BY BROMOENOL LACTONE AND TRIFLUOROMETHYL KETONES

A novel Ca2+-independent phospholipase A(2) (PLA(2)) has recently been purified from the murine macrophage-like cell line P388D(1) (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF(3)), arachidonyl trifluoromethyl ketone (AACOCF(3)), and a bromoenol lactone (EEL). Both PACOCF(3) and AACOCF(3) were found to inhibit the macrophage PLA(2) in a concentration-dependent manner, PACOCF(3) was found to be similar to 4-fold more potent than AACOCF(3), with IC50 values of 3.8 mu M (0.0075 mol fraction) and 15 mu M (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF(3).PLA(2) complex rapidly dissociated upon dilution. BEL was also found to inhibit the macrophage PLA(2) in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40 degrees C, Inhibition was not reversed after extensive dilution of the enzyme into assay buffer, Treatment of the PLA(2) with EEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF(3). In addition, PLA(2) treated with [H-3]BEL resulted in the covalent labeling of a major band at M(r) 80,000. Inactivation of the PLA(2) by 5,5'-dithiobis(2-nitrobenzoic acid) prior to treatment with [H-3]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a M(r) 80,000 band rather than a M(r) 40,000 band upon treatment with [H-3]BEL distinguishes the macrophage Ca2+-independent PLA(2) from a previously identified myocardial Ca2+-independent PLA(2) and provides strong evidence that the M(r) 80,000 protein is the catalytic subunit.