The size and curvature of anionic covesicle substrate affects the catalytic action of cytosolic phospholipase A2

Cytosolic phospholipase A(2) (cPLA(2)) is normally located in the cytosol, but in response to cellular activation the enzyme binds to the membrane at the lipid/water interface where it catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. Synthetic phospholipid vesicle systems have been used in kinetic and mechanistic analyses of cPLA(2), but these systems result in a rapid loss of enzyme activity. In the present research, covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing less than or equal to 10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) as substrate were used to show that this premature cessation of enzyme-catalyzed hydrolysis is dependent on vesicle size with 25-nm-diameter vesicles supporting little activity as compared to 100-, 200-, and 400-nm vesicles. This suggests that the curvature of the vesicle may shift a conformational equilibrium toward an enzyme state which does not support activity. Interestingly, the presence of 30% (v/v) glycerol greatly enhanced the activity of the enzyme, although vesicle size-dependent premature cessation of hydrolysis was still observed. While the premature cessation of hydrolysis in the absence of glycerol is accompanied by enzyme inactivation, little inactivation occured in the presence of glycerol, indicating that premature cessation and inactivation are not absolutely coupled. When using this covesicle substrate system under conditions (6-10 mM CaCl2) where the vesicles are fusing, no premature cessation of hydrolysis has been observed. This is despite a mean vesicle diameter of 400-450 nm under vesicle-fusing conditions, which is comparable to the largest vesicles used under nonfusing conditions (0.5 mM CaCl2) where considerable premature cessation of hydrolysis was observed. Since DMPM has an intrinsic active site dissociation constant at least 330 times larger than that of PAPC, the optimum conditions for conducting kinetic and mechanistic analyses of cPLA(2) with this covesicle substrate is one in which cPLA(2) is assayed in the presence of glycerol and with fusion-inducing concentrations of calcium. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (DOPM) instead of DMPM in this system supports much less activity and adds the complication of a strong affinity of DOPM for the active site. (C) 1999 Academic Press.