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 Cu
2+, Zn
2+, Hg
2+ and Cd
2+. The enzyme was activated by sulfhydryl compounds and Co
2+. 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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