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为了研究多催化功能蛋白酶（ｍｕｌｔｉｃａｔａｌｙｔｉｃａｌｐｒｏｔｅｉｎａｓｅ，ＭＣＰ）在负氮平衡形成中的作用，以大鼠骨骼肌为原料，提取此酶并制备其抗血清．将大鼠骨骼肌粗提物经４５％～６５％饱和度硫酸铵分级盐析、阴离子交换层析和凝胶过滤，最后从Ｓｅｐｈａｒｏｓｅ４Ｂ层析柱上获得单一活性洗脱峰．酶活性用Ｃａｒｂｏｂｅｎｚｏｘｙ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－４－ｎｉｔｒｉｎｉｌｉｄｅａｃｅｔａｔｅ作底物检测．非变性ＰＡＧＥ银盐染色显示单一区带的骨骼肌多催化功能蛋白酶，ＳＤＳ－ＰＡＧＥ银盐染色显示１０条亚基电泳区带，分子量在２５～３２ｋＤ之间．纯化的酶免疫兔８周后，抗血清效价达１３２，用分级盐析和离子交换层析纯化抗血清，显示单一电泳区带的ＩｇＧ．Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析显示只在２５～３２ｋＤ之间出现多条亚基区带．这些结果提示已获得电泳纯ＭＣＰ及其较高特异性的多克隆抗体．
In order to study the role of multicatalytical protease (MCP) in the formation of negative nitrogen balance, rat skeletal muscle was used as raw material to extract this enzyme and prepare its antisera. The rat skeletal muscle crude extracts were fractionated by ammonium sulfate (45% ~ 65%), anion exchange chromatography and gel filtration. Finally, a single active eluting peak was obtained from Sepharose 4B column. Enzyme activity was detected using Carbobenzoxy-Val-Gly-Arg-4-nitrinilideacetate as a substrate. Non-denaturing PAGE silver salt staining showed a single band of skeletal muscle multi-catalytic protease, SDS-PAGE silver salt staining showed 10 subunits electrophoresis zone, the molecular weight between 25 ~ 32kD. After 8 weeks of purification, the antiserum titer reached 132, and the antisera was purified by fractional salting-out and ion exchange chromatography. The IgG of the single electrophoresis zone was displayed. Western-blot analysis revealed multiple subunit bands between 25 and 32 kD. These results suggest that electrophoresed pure MCP and its more specific polyclonal antibodies have been obtained.