SEPARATION OF ACTIVE TUBULIN AND MICROTUBULE-ASSOCIATED PROTEINS BY ULTRACENTRIFUGATION AND ISOLATION OF A COMPONENT CAUSING THE FORMATION OF MICROTUBULE BUNDLES
SEPARATION OF ACTIVE TUBULIN AND MICROTUBULE-ASSOCIATED PROTEINS BY ULTRACENTRIFUGATION AND ISOLATION OF A COMPONENT CAUSING THE FORMATION OF MICROTUBULE BUNDLES
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DOI：
10.1021/bi00313a026
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
LIN, CM
中科院分区：
文献类型：
--
作者：
HAMEL, E;LIN, CM
建立了一种分离[小牛脑]微管相关蛋白（MAP）和微管蛋白的新方法，适合于相对大规模的制备。用1.6 m2 -(N-morpholino)乙磺酸盐（Mes）和GTP诱导微管蛋白选择性聚合后，经离心分离法分离出大部分活性微管蛋白。富集map的上清液浓缩，随后通过长时间离心澄清。上清液（总可溶性MAP）几乎不含微管蛋白，微管蛋白的核苷二磷酸激酶活性大部分，促进微管组装的活性良好，在0.1 M的Mes和具有MAP-1、MAP-2和.tau的电泳迁移率的蛋白。的因素。该颗粒不支持微管组装，含有变性微管蛋白，微管蛋白的大部分atp酶活性和大量具有MAP-2电泳迁移率的蛋白质。在这一阶段和之前所有阶段的不溶性物质，包括微管蛋白的制备，都可以被热提取，以产生可溶性蛋白，这些蛋白在促进微管组装方面具有活性，并以MAP-2为主要成分。总可溶性MAP通过deae -纤维素层析进一步纯化为结合组分和非结合组分，两者均诱导微管组装。结合组分（DEAE-MAP）含有具有MAP-1、MAP-2和.tau蛋白的电泳迁移率。的因素。由未结合组分（流过MAP）引起的聚合反应产生非常高的浊度读数。这是由微管束的形成引起的。尽管与DEAE-MAP相比，流动MAP含有更多的atp酶、小管蛋白无关的GTPase，尤其是核苷二磷酸激酶活性，但制备没有这些酶的MAP片段需要热处理。
A new method for separating [calf brain] microtubule-associated proteins (MAP) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAP by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAP-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAP) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtuble protein, good activity in promoting microtubule assembly in 0.1 M Mes and proteins with the electrophoretic mobility of MAP-1, MAP-2 and .tau. factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAP were further purified by DEAE-cellulose chromatogrpahy into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAP) contained proteins with the electrophoretic mobility of MAP-1, MAP-2 and .tau. factor. The polymerization reaction induced by the unbound component (flow-through MAP) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAP contained significantly more ATPase, tubulin-independent GTPase and, especially, nucleosidediphosphate kinase activity than the DEAE-MAP, preparation of a MAP fraction without these enzymes required heat treatment.