A Continuous Fluorescence-Based Assay for the Human High-Molecular-Weight Cytosolic Phospholipase A2

✍ Scribed by Z. Huang; F. Laliberte; N.M. Tremblay; P.K. Weech; I.P. Street

• Book ID: 102966247
• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 543 KB
• Volume: 222
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1461

✦ Synopsis

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase (A_{2}) (\left(\mathrm{cPLA}{2}\right)) ) is described. Recombinant cPLA (\mathbf{A}{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 (\mathrm{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 (\mathrm{cPL} \mathrm{A}{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 (A_{2}), also inhibited the 7-HCEase activity. A study of the 7HCEase activity demonstrated that when 7 -hydroxycoumarinyl (\gamma)-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by (\mathbf{c P L} \mathbf{A}{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 (\mathrm{cPLA} \mathbf{A}{2})-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The (\mathbf{c P L A} \mathbf{A}{2})-catalyzed hydrolysis of the water-soluble substrate 7 -hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA (A{2}) in the absence of calcium and other lipids. 1994 Academic Press, Inc.