Purification and characterization of human seminal plasma aminopeptidase.
A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.
D000409:Alanine / Q000031:analogs & derivatives; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D012661:Semen / Q000201:enzymology*; D013379:Substrate Specificity
C028104:alanine-beta-naphthylamide; D004492:Edetic Acid; D000626:Aminopeptidases; D000409:Alanine