Partial purification and some properties of three kinds of aminopeptidase from carp [Cyprinus carpio] muscle

From the ammonium sulfate precipitate fraction (50-80%) of the crude extract of carp muscle, three kinds of aminopeptidase (designated AP-Ia, AP-II and AP-III) with different substrate specificity were separated by DEAE-Sephacel, hydroxyapatite and Sephadex G-150 column chro-matography. The molecular weights of AP-Ia, AP-II and AP-III were estimated to be about 73, 000, 115, 000 and 107, 000 by gel filtration, respectively. The pH optima of each enzyme wereabout 7.3, 8.5, and 7.3, respectively. AP-Ia by.drolyzed amino acid-β-naphthylamides (Pro->Ala->Leu->Val->Phe-βNA), (Ala)2, (Ala)3, and (Ala)4. But Asp-βNA, Gly-βNA, Gly-Gly and Leu-Gly were resistant to hydrolysis. AP-II was strongly inactivated by p-chloromercuribenzoate (PCMB) and o-phenanthroline (ο-PHE), and slightly activated by Co2+, Mn2+ and Mg2+. A variety of dipeptide were hydrolyzed by this enzyme, but amino acid-β-naphthylamides, tripeptides, (Ala)4, dipeptide-amides and benzoyl- dipeptides were not hydrolyzed. AP-III was inactivated by PCMB and ο-PHE, and slightly activated by dithiothritol (DTT) and Co2+. Varlous tripeptides were hydrolyzed by this enzyme, but dipeptides, (Ala)4 and amino acid-β-naphthylamides were not hydrolyzed. From these results, AP-II and AP-III are probaly dipeptidase [EC 3.4.13.11] and tripeptide aminopeptidase [EC 3.4.11.4], respectively.