Purification and characterization of an aminopeptidase from bovine leukocytes.
An aminopeptidase was purified about 4,000-fold from the clarified homogenate of bovine leukocytes by a series of column chromatographies on DEAE-cellulose, hydroxyapatite, Sephadex G-150, and DEAE-Toyopearl. The purified enzyme had a specific activity of 3.8 mumol X min-1 X mg-1 with arginine beta-naphthylamide (Arg-2-NNap) as substrate, and a minute amount of contaminating protein was found to be present by gel electrophoresis. The molecular weight of the enzyme was estimated to be 94,000 by gel filtration on Sephadex G-150. The enzyme had a broad substrate specificity and a pH optimum between 6.5 and 7.0 for the hydrolysis of alpha-aminoacyl beta-naphthylamides. It hydrolyzed beta-naphthylamides of basic, aliphatic, and aromatic amino acids, and also catalyzed the liberation of amino-terminal phenylalanine from phenylalanyl peptides. The enzyme was inhibited by bestatin, puromycin, 1,10-phenanthroline, sulfhydryl reagents, and a variety of heavy metal ions. Only the cobaltous ion stimulated the enzyme and the values of both Km and Vmax for Arg-2-NNap increased. In gross properties the present enzyme resembles porcine liver aminopeptidase reported previously (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101) very closely.
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002627:Chemistry, Physical; D002845:Chromatography / Q000379:methods; D003035:Cobalt / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007962:Leukocytes / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity
D008670:Metals; D003035:Cobalt; D000626:Aminopeptidases