A continuous fluorescence-based assay for the human high-molecular-weight cytosolic phospholipase A2.
A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 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 cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, 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 cPLA2 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 cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.
Department of Biochemistry and Molecular Biology, Merck Forsst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Z
Z
Huang
F
F
Laliberté
N M
NM
Tremblay
P K
PK
Weech
I P
IP
Street
D002118:Calcium / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008245:Lysophospholipase / Q000302:isolation & purification; D008970:Molecular Weight; D010741:Phospholipases A / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2; D014468:Umbelliferones / Q000737:chemistry
D005456:Fluorescent Dyes; D014468:Umbelliferones; C031477:7-hydroxycoumarin; D010741:Phospholipases A; C091904:7-hydroxycoumarinyl ester hydrolase; D054467:Phospholipases A2; D008245:Lysophospholipase; D002118:Calcium