Institute of Food Processing, Zhejiang Academy of Agriculture Sciences, Hangzhou, 310021, China.
Fish Physiol Biochem. 2010 Dec;36(4):953-62. doi: 10.1007/s10695-009-9372-0. Epub 2009 Dec 19.
Aminopeptidases play important roles in turnover of proteins, metabolism of hormones and neurotransmission, cell maturation and immunological regulations. In the present study, an aminopeptidase was purified to homogeneity from the skeletal muscle of grass carp by ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite and Phenyl-Sepharose. The purified enzyme revealed a molecular mass of approximately 105 kDa both on SDS-PAGE and on gel filtration of Superdex 200. The enzymatic activity toward synthetic substrates was optimal at 40°C and pH 7.0-7.5. Metal-chelating agents such as EDTA and EGTA effectively inhibited the enzyme activity while inhibitors to serine, asparatic and cysteine proteinases did not show much effect, suggesting its belonging to metalloproteinase family. A specific aminopeptidase inhibitor bestatin was most effective in suppressing the enzymatic activity and performed in a competitive fashion. The enzymatic activity was slightly enhanced by metal ions of Mg2+ and Mn2+ while inhibited to different extents by Co2+, Cu2+, Zn2+ and Ca2+. Sulfhydryl reagent was necessary to maintain its activity. Purified enzyme demonstrated amidolytic activity most effectively against synthetic aminopeptidase substrate Leu-methylcoumarylamide (MCA) while N-terminal-blocked substrates and myofibrillar proteins were not hydrolyzed. The enzyme purified in the present study was quite possibly a leucine aminopeptidase (LAP) and functions during muscular protein metabolism.
氨肽酶在蛋白质的代谢、激素和神经递质的代谢、细胞成熟和免疫调节中起着重要作用。本研究采用硫酸铵分级沉淀和连续层析步骤(包括 DEAE-Sephacel、Sephacryl S-200、羟磷灰石和苯基-Sepharose),从草鱼骨骼肌中纯化出一种氨肽酶,达到了均一性。纯化的酶在 SDS-PAGE 和 Superdex 200 凝胶过滤中均显示出约 105 kDa 的分子量。该酶对合成底物的酶活性在 40°C 和 pH 7.0-7.5 时最佳。金属螯合剂如 EDTA 和 EGTA 可有效抑制酶活性,而丝氨酸、天冬氨酸和半胱氨酸蛋白酶抑制剂则没有太大影响,表明其属于金属蛋白酶家族。一种特定的氨肽酶抑制剂亮肽素对抑制酶活性最有效,呈竞争性抑制。Mg2+和 Mn2+等金属离子对酶活性有轻微增强作用,而 Co2+、Cu2+、Zn2+和 Ca2+则不同程度地抑制了酶活性。巯基试剂是维持其活性所必需的。纯化的酶对合成氨肽酶底物亮氨酸甲基香豆素酰胺(MCA)表现出最强的酰胺水解活性,而 N-末端封闭的底物和肌原纤维蛋白则不能被水解。本研究中纯化的酶很可能是亮氨酸氨肽酶(LAP),在肌肉蛋白代谢中发挥作用。