Continuous, Vesicle-Based Fluorimetric Assays of 14- and 85-kDa Phospholipases A2

Abstract

This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A2(cPLA2) and the 14-kDa, human nonpancreatic, secreted phospholipase A2(sPLA2). Phosphatidylcholines containing an arachidonyl chain at thesn-2 position and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at thesn-1 position were synthesized and shown to be substrates for cPLA2in 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 cPLA2assays 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 cPLA2. The ester formed between γ-linolenic acid and 7-hydroxycoumarin is also a substrate for cPLA2, and when incorporated into vesicles of the anionic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphomethanol, provides an assay in which the enzyme does not leave the vesicle surface (scooting mode). Unlike all of the previously reported, vesicle-based cPLA2assays, a prolonged linear reaction progress is seen with the DOPM-based assay. An assay of sPLA2with 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 sPLA2but provide a trap for enzyme-produced 10-pyrenedecanoic acid. The fluorescence of monomeric pyrene label in sink vesicles is detected. A second sPLA2assay 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 cPLA2and sPLA2assays are ideal for inhibitor screening and analysis, and for studying the interfacial kinetics of these enzymes.