Purification and Characterization of an Aminopeptidase from Porcine Liver

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Abstract
An aminopeptidase was purified about 700-fold from porcine liver homogenate by ammonium sulfate fractionation and a series of column chromatographies on DEAE-cellulose, Sephadex G-150, DEAE-Sepharose, and hydroxyapatite. The purified enzyme had a specific activity of 4.6 μmol .min−1.mg−1 using L-leucine βnaphthylamide as the substrate. The molecular weight of the enzyme was about 96,000 as determined by Sephadex G-150 column chromatography. The enzyme had a broad specificity and a pH optimum between 6.5 and 7.0 for hydrolysis of α-aminoacyl-β-naphthylamines, and it hydrolyzed the β-naphthylamides of aliphatic, basic, and aromatic amino acids. The enzyme also hydrolyzed peptide substrates with phenylalanine residues as their amino-termini, but it did not hydrolyze L-phenylalanyl-L-proline. The enzyme was inhibited by metal-chelating agents, sulfhydryl reagents, heavy metals, bestatin, and puromycin. Activity of the enzyme inhibited by sulfhydryl-reactive reagents was restored by the addition of sulfhydryl compounds. The enzyme was activated by cobaltous ion and the values of both Km and Vmax increased. The activation was pH-dependent and above pH 7.5 cobalt ion behaved as an inhibitor of the enzyme. No metal ions other than cobaltous ion stimulated the enzyme appreciably.