A CONTINUOUS FLUORESCENCE-BASED ASSAY FOR THE HUMAN HIGH-MOLECULAR-WEIGHT CYTOSOLIC PHOSPHOLIPASE A(2)

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A(2) (cPLA(2)) is described. Recombinant cPLA(2) efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA(2) was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA(2) by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A(2) and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A(2) activity of cPLA(2), also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl gamma-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA(2) at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA(2)-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA(2)-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA(2) in the absence of calcium and other lipids. (C) 1994 Academic Press, Inc.