Y. Kim et al., A PHOSPHOLIPASE A(2) KINETIC AND BINDING ASSAY USING PHOSPHOLIPID-COATED HYDROPHOBIC BEADS, Analytical biochemistry, 250(1), 1997, pp. 109-116
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.