Purification and properties of phospholipase A 2 from human seminal plasma

Abstract

The soluble Ca 2+-dependent phospholipase A 2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates ( V max = 494 U/mg, and K m , = 1.25 · 10 −4 M long-chain phosphatidylethanolamine; V max = 7.4 U/ mg, and K m = 2.5 · 10 −5 M long-chain phosphatidylcholine; V max = 7196 U/ mg and K m = 8.32 · 10 −4 M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A 2, no immunochemical relationship was observed; anti-pancreatic phospholipase A 2 IgGs did not inhibit seminal phospholipase A 2. Similarly, partially purified phospholipase A 2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A 2.