Partial Purification and Some Properties of Three Kinds of Aminopeptidase from Carp Muscle

Abstract

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.