College of Environmental and Chemical Engineering, Nanchang Hangkong University, Nanchang, China.
Comp Biochem Physiol B Biochem Mol Biol. 2011 May;159(1):10-7. doi: 10.1016/j.cbpb.2011.01.005. Epub 2011 Jan 27.
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.
一种来自斑马鱼（Danio rerio）的氨肽酶通过依次在 DEAE-葡聚糖、羟磷灰石和苯琼脂糖上进行柱层析，以 35.4%的回收率纯化了 1247 倍至均一性。该酶的分子量通过 SDS-PAGE 和凝胶过滤估计为 98 kDa。该酶的最适温度和 pH 分别为 45°C 和 7.5。该酶优先水解 Leu-MCA 作为底物，k(cat)/K(m)为 4.2×10(6)M(-1)s(-1)，活化能为 68.9 kJ M(-1)[已更正]。它被 bestatin、puromycin 和金属螯合剂特异性抑制，并且 Zn(2+)似乎是其金属辅因子。一些 l-氨基酸显著抑制其活性，而 l-半胱氨酸是一种非竞争性抑制剂，K(i)为 0.27 mM。根据肽质量指纹图谱分析，该酶与预测的 D. rerio 氨肽酶 puromycin 敏感型（gi：255683530）（EC 3.4.11.14）高度匹配，表明该酶是斑马鱼的 puromycin 敏感型氨肽酶。
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