Irreversible inhibition of Ca2+-independent phospholipase A(2) by methyl arachidonyl fluorophosphonate

Methyl arachidonyl fluorophosphonate (MAFP) has been recently reported to be a selective, active-site directed, irreversible inhibitor of the Group IV 85 kDa cytosolic phospholipase A(2) (c:PLA(2)). We have now shown that this compound also potently inhibits the Ca2+-independent cytosolic phospholipase A(2) (iPLA(2)). MAFP inhibited IPLA(2) in a concentration-dependent manner with half-maximal inhibition observed at 0.5 mu M after a 5 min preincubation at 40 degrees C. This inhibition was not reversed upon extensive dilution of the enzyme into the assay mixture. Preincubation of iPLA(2) with MAFP resulted in a linear, time-dependent inactivation of enzyme activity, and the enzyme was protected from inactivation by the reversible inhibitor PACOCF(3). The ability of MAFP to inhibit the iPLA(2) suggests that this enzyme proceeds through an acyl-enzyme intermediate as has been proposed for the cPLA(2). Further testing indicated that MAFP did not inhibit the arachidonoyl-CoA synthetase, CoA-dependent acyltransferase, or CoA-independent transacylase activities from P388D(1) cells. Thus, MAFP is not a general inhibitor for enzymes which act on arachidonoyl substrates. Instead, the inhibitor appears to show some selectivity for PLA(2), although it does not discriminate between cPLA(2) and iPLA(2). Particular caution must be exercised to distinguish these activities if this inhibitor is used in intact cells.