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)). Phosphatidylcholines containing an arachidonyl chain at the sn-2 position 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 large unilamellar vesicles of substrate phospholipid, although the assay also works when the substrate is dispersed in Triton X-100 mixed-micelles. 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 continuous when bovine serum albumin is present in the aqueous phase; this protein extracts the pyrene-containing lysophospholipid from the vesicle, and this leads to the fluorescence of monomeric pyrene label. These assays are capable of detecting subnanogram amounts of cPLA(2). The ester formed between gamma-linolenic acid and 7-hydroxycoumarin is also a substrate for cPLA(2), and when incorporated into vesicles of the anionic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphomethano provides an assay in which the enzyme does not leave the vesicle surface (scooting mode). Unlike all of the previously reported, vesicle-based cPLA(2) assays, a prolonged linear reaction progress is seen with the DOPM-based assay. An assay of sPLA(2) with subnanogram sensitivity was developed which makes use of the substrate 1-palmitoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphomethanol and a lipid sink. The latter is composed of phosphatidylcholine vesicles, in excess of substrate vesicles, which do not bind sPLA(2) but provide a trap for enzyme-produced 10-pyrenedecanoic acid. The fluorescence of monomeric pyrene label in sink vesicles is detected. A second sPLA(2) assay using a single type of vesicle was developed based on the substrate 1,2-di(10-pyrenedecanoyl)-sn-glycero-3-phosphocholine present at 10 mol% in vesicles of the nonhydrolyzable anionic phospholipid 1,2-ditetradecyl-sn-glycero-3 -phosphomethanol. The action 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.