Purification and characterization of rabbit platelet cytosolic phospholipase A2.

A phospholipase A2 was purified from rabbit platelet cytosolic fraction to near homogeneity by sequential column chromatographies on heparin-Sepharose, DEAE-Sephacel, butyl-Toyopearl, DEAE-5PW ion-exchange HPLC, and TSK gel G3000SW gel-filtration HPLC. The final preparation with an estimated specific activity of 8630 nmol/min per mg protein, showed a single band with a molecular mass of about 88 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The 88-kDa phospholipase A2 exhibited a fatty acid preference; it hydrolyzed phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than that with a linoleoyl residue. The catalytic activity of the purified enzyme with phosphatidylcholine or phosphatidylethanolamine increased sharply in the presence of between 10(-7) and 10(-6) M calcium ion, indicating that it could be regulated by less than micromolar concentration of calcium. These characteristics differ from those of platelet secretory 14-kDa phospholipase A2 reported previously. Therefore, this 88-kDa enzyme is a novel phospholipase A2 and may participate in the stimulus-dependent release of arachidonoyl residues in rabbit platelets.