PROTEIN-KINASE ASSOCIATED WITH TUBULIN - AFFINITY CHROMATOGRAPHY AND PROPERTIES
PROTEIN-KINASE ASSOCIATED WITH TUBULIN - AFFINITY CHROMATOGRAPHY AND PROPERTIES
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DOI：
10.1021/bi00661a005
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
CUATRECASAS, P
中科院分区：
文献类型：
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作者：
SANDOVAL, IV;CUATRECASAS, P
用秋水仙素-琼脂糖亲和层析法纯化的大鼠脑微管蛋白具有蛋白激酶活性。通过酪蛋白柱层析将激酶活性与微管蛋白完全分离，并且随后不被秋水仙素亲和柱保留。与纯化的微管蛋白相关的蛋白激酶活性与脑匀浆中蛋白激酶活性的总含量无关，因为从48，000 g胎脑上清液中分离的微管含有比成人微管多两倍的蛋白激酶活性，尽管总蛋白激酶活性是48，000 g成人上清液中的两倍。微管蛋白制剂中的蛋白激酶虽然对应于与微管蛋白不同的分子，但可能不仅仅是污染的结果。这些观察结果被解释为蛋白激酶和微管蛋白复合物之间的特定关联。蛋白激酶与微管蛋白的结合可能是调节微管蛋白功能的重要决定因素。在相同微管蛋白浓度下，在不存在甘油的情况下，胎儿微管蛋白聚合两倍于成人微管蛋白。体内或体外蛋白激酶的优选底物（pH7.4，37 ℃）是：C）是一种特异的高分子量蛋白质，不同于微管蛋白，其通过不同种类的分离程序与微管蛋白共纯化（即，秋水仙碱亲和层析和（NH 4）2SO 4沉淀，随后进行二乙基氨基乙基纤维素层析）。微管蛋白相关蛋白激酶完全依赖于环[c]AMP（Km = 10-7 M），如加入蛋白激酶调节剂（一种众所周知的cAMP依赖性蛋白激酶特异性抑制剂）后活性完全抑制所示。
Rat brain tubulin purified by colchicine-agarose affinity chromatography contains protein kinase activity. The kinase activity was separated completely from tubulin by chromatography on casein columns and was not subsequently retained by colchicine affinity columns. Protein kinase activity associated with purified tubulin did not correlate with the total content of protein kinase activity in brain homogenates, since microtubules isolated from 48,000 g fetal brain supernatants contained twice as much protein kinase activity than adult microtubules, although the total protein kinase activity was twice as high in the 48,000 g adult supernatant. The protein kinase of tubulin preparations, while corresponding to a different molecule than tubulin, was probably not simply the result of contamination. These observations were interpreted in terms of specific associations between protein kinase and tubulin complexes. The protein kinase-tubulin association may be an important determinant in the regulation of tubulin function. Fetal tubulin polymerized twice as well as adult tubulin in the absence of glycerol at the same tubulin concentration. The preferred substrate for the protein kinase either in vivo or in vitro (pH 7.4, 37.degree. C) was a specific high-molecular-weight protein, distinct from tubulin, which copurified with tubulin through different kinds of isolation procedures (i.e., colchicine affinity chromatography and (NH4)2SO4 precipitation followed by diethylaminoethyl-cellulose chromatography). The tubulin-associated protein kinase was completely dependent on cyclic[c]AMP (Km = 10-7 M), as demonstrated by the complete suppression of activity upon addition of the protein kinase modulator, a well-known specific inhibitor of cAMP-dependent protein kinases.