title: Identification of a puromycin-sensitive aminopeptidase from zebrafish (Danio rerio).
abstract: An aminopeptidase from zebrafish (Danio rerio) was purified 1247-fold to homogeneity with 35.4% recovery by column chromatography successively on DEAE-sephacel, hydroxyapatite, and phenyl-sepharose. The molecular mass of the enzyme was estimated at 98 kDa by SDS-PAGE and gel filtration. Optimum temperature and pH of the enzyme were 45°C and 7.5, respectively. The enzyme preferentially hydrolyzed substrate Leu-MCA with k(cat)/K(m) of 4.2×10(6)M(-1)s(-1) and an activation energy of 68.9 kJ M(-1) [corrected], respectively. It was specifically inhibited by bestatin, puromycin and metal-chelating agents, and Zn(2+) seemed to be its metal cofactor(s). Some l-amino acids significantly inhibited its activity, and l-cysteine was a non-competitive inhibitor with a K(i) of 0.27 mM. According to the peptide mass fingerprint analysis, the enzyme was highly matched with the predicted D. rerio aminopeptidase puromycin sensitive (gi: 255683530) (EC 3.4.11.14), suggesting that the present enzyme is a puromycin-sensitive aminopeptidase of zebrafish.
authors.affiliation: College of Environmental and Chemical Engineering, Nanchang Hangkong University, Nanchang, China.
authors.forename: Su-Hua
authors.initials: SH
authors.lastname: Chen
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
authors.forename: Jian-Zhen
authors.initials: JZ
authors.lastname: Huang
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D013379:Substrate Specificity; D029961:Zebrafish Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011480:Protease Inhibitors; D029961:Zebrafish Proteins; D011691:Puromycin; D000626:Aminopeptidases