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 styrene-divinylbenzene beads (5.2 +/- 0.3 mu m diameter). Phospholipids formed a stable monolayer film on styrene-divinylbenzene beads with average surface packing density of (1.3 +/- 0.2) x 10(-2) molecule/Angstrom(2). Secretory PLA(2) readily hydrolyzed 1-palmitoyl-2-[H-3]-oleoyl-sn-glycero-3-phosphoglycerol coated on styrene-divinylbenzene beads which could be easily monitored by measuring the radioactivity of fatty acid released to solution in the presence of bovine serum albumin. For human cytosolic PLA(2) with high specificity for sn-2 arachidonyl group, styrene-divinylbenzene beads coated with 1-stearoyl-2- [C-14]-arachidonyl-sn-glycero-3-phosphocholine and dioleoylglycerol (7:3, mol/mol) were used as substrate. PLA(2) activity was linearly proportional to the enzyme concentration in the range from 1 to 150 nM for human class II secretory 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, styrene-divinylbenzene beads coated with polymerized 1,2-bis[12-(lipoyloxy) dodecanoyl]-sn-glycero-3-phosphoglycerol were used to measure the membrane binding affinity of PLA(2), which in conjunction with kinetic data provides important insights into how PLA(2) interacts with membranes. (C) 1997 Academic Press.