A CONTINUOUS FLUORESCENCE-BASED ASSAY FOR THE HUMAN HIGH-MOLECULAR-WEIGHT CYTOSOLIC PHOSPHOLIPASE A2

Abstract 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 γ-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.