PURIFICATION AND CHARACTERIZATION OF A MULTICATALYTIC HIGH-MOLECULAR-MASS PROTEINASE FROM RAT SKELETAL-MUSCLE
PURIFICATION AND CHARACTERIZATION OF A MULTICATALYTIC HIGH-MOLECULAR-MASS PROTEINASE FROM RAT SKELETAL-MUSCLE
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DOI：
10.1042/bj2280161
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发表时间：
1985-01-01
影响因子：
4.1
通讯作者：
REINAUER, H
中科院分区：
文献类型：
--
作者：
DAHLMANN, B;KUEHN, L;REINAUER, H
从大鼠骨骼肌肌原纤维后部分提纯了一种蛋白水解酶。肌肉提取物经(NH4)2SO4分级、DEAE-Sephacel柱层析、Mono Q快速蛋白层析和Sepharose6 B凝胶过滤后，经聚丙烯酰胺凝胶圆盘电泳法和免疫电泳法鉴定为均一酶制剂。该酶的等电点为5.1-5.2。这种酶的分子量为.apprx。在十二烷基硫酸钠/聚丙烯酰胺凝胶中进行电泳时，可解离为8个相对分子质量为25,000-32,000的亚基。该酶对N-封闭的三肽4-甲基-7-香豆酰胺底物具有水解性，离开基团附近有精氨酸或苯丙氨酸残基。第一组底物的最大活性是在pH 10.5时，这一活性被亮肽素、糜抑素和钙离子抑制。后一组底物的最大活性在pH 7.5时，也被两种微生物抑制剂抑制，但被钙激活。以[~(14)C]甲基酪蛋白为底物，在pH为9.0时有最大活性，这种活性不受亮氨酸蛋白的影响，被糜抑素增强，被钙离子抑制。以苄氧羰基-亮氨酸-谷氨酸-2-萘胺为底物时，也观察到了类似的效果。这些酶的活性可被对羟基汞苯磺酸或Mersalyl所消除，而被半胱氨酸或二硫苏糖醇所激活。
A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6 B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has a MW of .apprx. 650,000 and dissociates into 8 subunits of MW 25,000-32,000 when subjected to electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the 1st group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the 2 microbial inhibitors, but was activated by Ca2+. By using [14C]methylcasein as a substrate, maximum activity was observed at pH 9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulfonic acid or mersalyl, whereas a small activation was observed with cysteine or dithiothreitol.