Purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

文献类型： 外文期刊

第一作者： Liu, Bing-Xin

作者： Liu, Bing-Xin;Du, Xue-Li;Su, Wen-Jin;Cao, Min-Jie;Zhou, Li-Gen;Hara, Kenji

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关键词： common carp;purification;leucine aminopeptidase

期刊名称：FOOD CHEMISTRY （ 影响因子：7.514； 五年影响因子：7.516 ）

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收录情况： SCI

摘要： An aminopeptidase was purified from the skeletal muscle of common carp (Cyprinus carpio) to homogeneity, with 1160-fold purification and a yield of 4.3%. The purification procedure consisted of ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, Phenyl Sepharose and Macro-Prep High Q columns. The molecular mass of the enzyme was estimated to be 105 kDa and 100 kDa by SDS-PAGE under reducing conditions and gel filtration chromatog-raphy, respectively, suggesting it to be a monomer. The enzymatic activity was optimal at 35℃ and pH 7.0. The metal-chelating agents EDTA, EGTA and 1,10-phenanthroline specifically inhibited the enzyme activity while inhibitors of other proteinases did not show much effect, indicating that it was a metalloproteinase. Furthermore, bestatin, a specific aminopeptidase inhibitor strongly inhibited its activity. Divalent cations Mn~(2+), Mg~(2+) and Ba~(2+) slightly enhanced the enzymatic activity, while Co~(2+), Cu~(2+), Zn~(2+), Ca~(2+) and Fe~(2+) inhibited the activity to different extents. In addition, a sulfhydryl reagent was necessary to maintain its activity. Substrate specificity study revealed that the purified enzyme preferentially hydrolyzed Leu-MCA, followed by Arg-MCA, Ala-MCA and Tyr-MCA and it was thus regarded as a leucine aminopeptidase (LAP, EC 3.4.11.1). The apparent K_m and V_(max) values of the enzyme were 4.6 μM and 9.6 μmoL min~(-1) mg~(-1), respectively for substrate Leu-MCA. This is the first report concerning purification and characterization of LAP from freshwater fish.

分类号： TS201`TS264`TS201.2

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