A leucine aminopeptidase was purified for the first time from marine fish red sea bream ( Pagrus major) skeletal muscle to homogeneity with 4850-fold and a yield of 7.4%. The purification procedure consisted of ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, and phenyl-Sepharose. The enzyme was approximately 96 kDa as estimated by SDS-PAGE and gel filtration and preferentially hydrolyzed substrate Leu-MCA. The enzymatic activity was optimal at 45 degrees C and pH 7.5. The K m and k cat values of the enzyme for Leu-MCA were 1.55 microM and 26.4 S (-1) at 37 degrees C, respectively. Activation energy ( E a) of the enzyme was 59.6 kJ M (-1). The enzyme was specifically inhibited by metal-chelating agents, and Zn (2+) and (or) Mn (2+) seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K i was 1.44 microM. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.

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