Purification and some properties of aminopeptidase from carp Cyprinus carpio ordinary muscle.

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Abstract
An aminopeptidase from carp Cyprinus carpio ordinary muscle was purified 700-fold by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and hydroxyapatite chromatography. The final enzyme preparation showed a single protein band on polyacrylamide gel electrophoresis (PAGE) and on sodium dodecyl sulfate (SDS) PAGE in the presence of 2-mercaptoethanol. The molecular weight of the enzyme was about 84, 000 and 94, 000 by gel filtration and SDS-PAGE, respectively. The enzyme was inhibited by p-chloromercuribenzoate, 5, 5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, p-tosyl-L-phenylalanine chloromethylketone, ptosyl-L-lysine chloromethylketone, EDTA, o-phenanthroline, puromycin and some heavy metal ions such as Cu2+, Zn2+, Hg2+ and Cd2+. The enzyme was activated by sulfhydryl compounds and Co2+. The enzyme had a broad specificity for hydrolysis of aminoacyl-β-naphthylamides (βNA), and pH optimum was 7.5. Ala-, Phe-, Lys-, Arg-, Leu-, Tyr- and Trp-βNA were suitable substrate for the enzyme, and Ser-, Pro-, Gly-, Val-, Ile- and Asp-βNA were poor substrate. Among the peptides used (Ala)5 and (Ala), were most rapidly hydrolyzed, and the relative rates of hydrolysis for both substrates were 2-fold higher than that of (Ala)3; and (Ala)3 was hydrolyzed about 60-fold higher than that of (Ala)2.

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