title: Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution.
abstract: 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.
authors.affiliation: School of Life Sciences, Xiamen University, Xiamen, China 361005.
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Wu
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Bing-Xin
authors.initials: BX
authors.lastname: Liu
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
mesh_terms: D000818:Animals; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology*; D010473:Perciformes / Q000378:metabolism*; D013379:Substrate Specificity; D014018:Tissue Distribution
chemical_list: D029941:Fish Proteins; D007931:Leucyl Aminopeptidase