大鼠骨骼肌多催化功能蛋白酶的提取、鉴定及其抗血清的制备

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．

Purification,Identification of the Multicatalytical Proteinase from Rat Skeletal Muscle

The MCP was extracted from rat skeletal muscle and the antisera to the MCP were raised for researching the role of the MCP during the formation of the negative nitrogen balance.Rat skeletal muscle was homogenized and centrifuged,then fractionated by 45%_65% saturation of (NH 4) 2SO 4,the pellet was chromatographed on DE 52 anion exchanger,the enzyme active protein liquid was then chromatographed on Sephadex 150 and Sepharose 4B respectively.Finally,single active eluting peak was obtained.Subjected to polyacrylamide gel electrophoresis under non denaturating conditions,the enzyme migrated as single band and under the conditions of SDS polyacrylamide gel electrophoresis,it depolymerized into ten subunits of M r 32_25 kD.The N blocked tripeptide Carbobenzoxy Val Gly Arg 4 nitrinilide acetate was used to detect the enzyme activity.The purified enzyme was mixed with Freund's complete adjuvant,and then administered subcutaneously for rabbits.Eight weeks later,the value of antisera reached 1∶32.The IgG form was then purified by fractionation with (NH 4) 2SO 4 and anion exchange column chromatography.The purified enzyme showed single spot in electrophoresis.Only between 25-32 kD,several subunits bands could be observed when the MCP was applied for Western blot analysis.These results suggested that the MCP of electrophoresising purity and high specific polyantibody had been acquired.