A PHOSPHOLIPASE A(2) KINETIC AND BINDING ASSAY USING PHOSPHOLIPID-COATED HYDROPHOBIC BEADS

A novel kinetic and membrane-binding assay for phospholipase A(2) (PLA (2)) has been developed utilizing phospholipid-coated hydrophobic styr ene-divinylbenzene beads (5.2 +/- 0.3 mu m diameter). Phospholipids fo rmed a stable monolayer film on styrene-divinylbenzene beads with aver age surface packing density of (1.3 +/- 0.2) x 10(-2) molecule/Angstro m(2). Secretory PLA(2) readily hydrolyzed mitoyl-2-[H-3]-oleoyl-sn-gly cero-3-phosphoglycerol coated on styrene-divinylbenzene beads which co uld be easily monitored by measuring the radioactivity of fatty acid r eleased to solution in the presence of bovine serum albumin. For human cytosolic PLA(2) with high specificity for sn-2 arachidonyl group, st yrene-divinylbenzene beads coated with 1-stearoyl-2- [C-14]-arachidony l-sn-glycero-3-phosphocholine and dioleoylglycerol (7:3, mol/mol) were used as substrate. PLA(2) activity was linearly proportional to the e nzyme concentration in the range from 1 to 150 nM for human class II s ecretory PLA(2) and from 1 to 20 nM for cytosolic PLA(2); the specific activity was 1.6 and 1.7 mu mol/min/mg, respectively. Finally, styren e-divinylbenzene beads coated with polymerized 1,2-bis[12-(lipoyloxy) dodecanoyl]-sn-glycero-3-phosphoglycerol were used to measure the memb rane binding affinity of PLA(2), which in conjunction with kinetic dat a provides important insights into how PLA(2) interacts with membranes . (C) 1997 Academic Press.