Leucine Aminopeptidase from Red Sea Bream (Pagrus major) Skeletal Muscle: Purification, Characterization, Cellular Location, and Tissue Distribution
Cao, Min-Jie（Biol Engn, Xiamen）
Liu, Bing-Xin（Biol Engn, Xiamen）
Chen, Yan（Jimei Univ）
- 生命科学－已发表论文 
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 SIDS-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 mu M 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 Zn2+ and (or) Mn2+ seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K-i was 1.44 mu M. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.