Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma.
A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.
D000176:Acrosin / Q000494:pharmacology; D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D012661:Semen / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000032:analysis / Q000302:isolation & purification*
D002250:Carbon Radioisotopes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D004492:Edetic Acid; D010738:Type C Phospholipases; D000176:Acrosin; D014357:Trypsin