COOPERATIVITY AND BINDING IN THE MECHANISM OF CYTOSOLIC PHOSPHOLIPASEA(2)

CytosoIlc phospholipase A(2) (cPLA(2)) hydrolyzes the sn-2 ester of ph ospholipids and is believed to be responsible for the receptor-regulat ed release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipi d substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PA PC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We rep ort here that the enzyme shows an apparent cooperative effect with res pect to the mole fraction of arachidonate-containing phospholipids wit hin these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phos phoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fu sion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosph atidylethanolamine (PAPE) does not show the same cooperative effect, a lthough the rate at which it is hydrolyzed is much greater when PAPC i s present. Moreover, PAPE has a dissociation constant from the active site (K-D = 0.7 mol %) which is comparable to that of PAPC and SAPI ( K-D values of 0.3 and 0.3 mol %, respectively). These results are con sistent with the presence of an allosteric site that, when occupied, i nduces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allos teric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilaye r concentration of arachidonate-containing phospholipids. This previou sly unobserved effect may represent another mechanism by which cells c an regulate the activity of cPLA(2).