MICROTUBULE ASSEMBLY INVITRO - PURIFICATION OF ASSEMBLY-PROMOTING FACTORS
MICROTUBULE ASSEMBLY INVITRO - PURIFICATION OF ASSEMBLY-PROMOTING FACTORS
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DOI：
10.1111/j.1432-1033.1977.tb11726.x
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发表时间：
1977-01-01
期刊：
影响因子：
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通讯作者：
NUNEZ, J
中科院分区：
文献类型：
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作者：
FELLOUS, A;FRANCON, J;NUNEZ, J
研究了[大鼠脑]微管相关蛋白在体外微管蛋白与微管组装中的作用。在缺乏与微管蛋白共聚合的次要成分的情况下，通过磷纤维素柱层析获得的纯微管蛋白在体外不能明显组装。虽然微管蛋白聚集在含有高浓度Mg2+的morpholino-乙磺酸缓冲液中，但这一过程既不会被Ca2+或秋水仙碱抑制，也不会被冷暴露逆转。采用磷纤维素柱层析法或在2 mM二硫苏糖醇和0.75 M NaCl存在下，用沸腾重组微管的直接方法制备了微管相关蛋白。通过超微凝胶ACA34层析或蔗糖梯度超离心，从每种制剂中纯化2个蛋白质组分。高分子量的第1馏分对微管蛋白组装没有促进作用；该材料的老化不产生任何活性。第二馏分的表观MW为70000 （.tau）。蛋白质)，当几乎完全纯化时，在促进组装方面是活跃的。因此，单一的特异性蛋白能够促进纯微管蛋白的组装。
The role of [rat brain] microtubule-associated proteins in the assembly of tubulin to microtubules in vitro was studied. Pure tubulin obtained by phosphocellulose collumn chromatography does not significantly assemble in vitro in the absence of minor components which co-polymerize with tubulin. Although tubulin aggregates in a morpholino-ethanesulfonate buffer containing high Mg2+ concentrations, this process was neither inhibited by Ca2+ or colchicine, nor reversed by cold exposure. Microtubule-associated proteins were prepared, either by phosphocellulose column chromatography or by a direct method based on boiling reassembled microtubules in the presence of 2 mM dithiothreitol and 0.75 M NaCl. From each of these preparations 2 protein fractions were purified, either by Ultrogel ACA34 chromatography or by sucrose gradient ultracentrifugation. The 1st fraction, with a high MW did not promote tubulin assembly; aging of this material did not induce any activity. The 2nd fraction with an apparent MW of 70,000 (.tau. protein), when almost completely purified, was active in promoting assembly. Thus a single specific protein is able to promote assembly of pure tubulin.