Identification of a puromycin-sensitive aminopeptidase from zebrafish (Danio rerio)

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 kcat/Km of 4.2 × 106 M−1 s−1 and an activation energy of 80.5 kJ M−1, respectively. It was specifically inhibited by bestatin, puromycin and metal-chelating agents, and Zn2+ 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 Ki 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.