| Abstract: | The soluble Ca2+-dependent phospholipase A2 (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 (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 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 A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2. |
