The predominant protein of canine seminal plasma is an enzyme.
One protein in canine seminal plasma accounts for over 90% of the total protein and is present at the high concentration of approximately 10 mg/ml. We demonstrate that this predominant protein is a proteolytic enzyme. The enzyme has been purified and migrates as a single symmetrical peak of apparent molecular mass of 29,000 daltons on a column of Sephadex G-75 and as a single band of approximately 30,000 daltons when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the enzyme dissociates into subunits of 15,000 and 12,000-14,000 daltons. The 15,000-dalton subunit contains the enzyme active site as determined by labeling with [3H]diisopropyl fluorophosphate. The enzyme hydrolyzed the synthetic ester substrates N alpha-benzoyl-L-arginine ethyl ester and N alpha-tosyl-L-arginine methyl ester with maximum specific activities at 25 degrees C of 105 mumol/min/mg and 33 mumol/min/mg, and Km values of 7.4 and 9.1 mM, respectively. The enzyme exhibited a pH optimum of 8.0. The metal ions, Cu2+, Zn2+, Cd2+, and Co2+ were reversible inhibitors and diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride irreversible inhibitors of enzymatic activity. By immunofluorescence, the enzyme can be detected on the tail and postacrosomal regions of washed ejaculated canine sperm, but it is absent from epididymal sperm.
D000177:Acrosome / Q000201:enzymology; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis; D007531:Isoflurophate / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000494:pharmacology; D012661:Semen / Q000201:enzymology*; D013082:Sperm Tail / Q000201:enzymology; D013379:Substrate Specificity
D002413:Cations, Divalent; D011480:Protease Inhibitors; D007531:Isoflurophate; D010447:Peptide Hydrolases