CONTINUOUS, VESICLE-BASED FLUOROMETRIC ASSAYS OF 14- AND 85-KDA PHOSPHOLIPASES A(2)

This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A(2) (cPLA(2)) and the 14-kDa, human nonpancreatic, secreted phospholipase A(2) (sPLA(2)). Ph osphatidylcholines containing an arachidonyl chain at the sn-2 positio n and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at the sn-1 position were synthesized and shown to be substrates for cPLA(2) in a fluorescence-based assay. Most of the assays make use of small and la rge unilamellar vesicles of substrate phospholipid, although the assay also works when the substrate is dispersed in Triton X-100 mixed-mice lles. The cPLA(2) assays can be carried out in a fixed time-point mode in which one of the products, the pyrene-containing lysophospholipid, is detected by rapid HPLC. Alternatively, the assay becomes continuou s when bovine serum albumin is present in the aqueous phase; this prot ein extracts the pyrene-containing lysophospholipid from the vesicle, and this leads to the fluorescence of monomeric pyrene label. These as says are capable of detecting subnanogram amounts of cPLA(2). The este r formed between gamma-linolenic acid and 7-hydroxycoumarin is also a substrate for cPLA(2), and when incorporated into vesicles of the anio nic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphomethano provides an assay in which the enzyme does not leave the vesicle surface (scootin g mode). Unlike all of the previously reported, vesicle-based cPLA(2) assays, a prolonged linear reaction progress is seen with the DOPM-bas ed assay. An assay of sPLA(2) with subnanogram sensitivity was develop ed which makes use of the substrate 2-(10-pyrenedecanoyl)-sn-glycero-3 -phosphomethanol and a lipid sink. The latter is composed of phosphati dylcholine vesicles, in excess of substrate vesicles, which do not bin d sPLA(2) but provide a trap for enzyme-produced 10-pyrenedecanoic aci d. The fluorescence of monomeric pyrene label in sink vesicles is dete cted. A second sPLA(2) assay using a single type of vesicle was develo ped based on the substrate -di(10-pyrenedecanoyl)-sn-glycero-3-phospho choline present at 10 mol% in vesicles of the nonhydrolyzable anionic phospholipid 1,2-ditetradecyl-sn-glycero-3 -phosphomethanol. The actio n of sPLA2 on this fluorescent substrate leads to a separation of the pyrene chains resulting in fluorescence emission from monomeric pyrene . These cPLA(2) and sPLA, assays are ideal for inhibitor screening and analysis, and for studying the interfacial kinetics of these enzymes. (C) 1995 Academic Press, Inc.