Purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

Abstract An aminopeptidase was purified from the skeletal muscle of common carp ( Cyprinus carpio ) to homogeneity, with 1160-fold purification and a yield of 4.3%. The purification procedure consisted of ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, Phenyl Sepharose and Macro-Prep High Q columns. The molecular mass of the enzyme was estimated to be 105 kDa and 100 kDa by SDS-PAGE under reducing conditions and gel filtration chromatography, respectively, suggesting it to be a monomer. The enzymatic activity was optimal at 35 °C and pH 7.0. The metal-chelating agents EDTA, EGTA and 1,10-phenanthroline specifically inhibited the enzyme activity while inhibitors of other proteinases did not show much effect, indicating that it was a metalloproteinase. Furthermore, bestatin, a specific aminopeptidase inhibitor strongly inhibited its activity. Divalent cations Mn 2+ , Mg 2+ and Ba 2+ slightly enhanced the enzymatic activity, while Co 2+ , Cu 2+ , Zn 2+ , Ca 2+ and Fe 2+ inhibited the activity to different extents. In addition, a sulfhydryl reagent was necessary to maintain its activity. Substrate specificity study revealed that the purified enzyme preferentially hydrolyzed Leu-MCA, followed by Arg-MCA, Ala-MCA and Tyr-MCA and it was thus regarded as a leucine aminopeptidase (LAP, EC 3.4.11.1). The apparent K m and V max values of the enzyme were 4.6 μM and 9.6 μmol min −1  mg −1 , respectively for substrate Leu-MCA. This is the first report concerning purification and characterization of LAP from freshwater fish.