COMPLETE SEPARATION OF TYROSINATED, DETYROSINATED, AND NONTYROSINATABLE BRAIN TUBULIN SUBPOPULATIONS USING AFFINITY-CHROMATOGRAPHY
COMPLETE SEPARATION OF TYROSINATED, DETYROSINATED, AND NONTYROSINATABLE BRAIN TUBULIN SUBPOPULATIONS USING AFFINITY-CHROMATOGRAPHY
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DOI：
10.1021/bi00432a050
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发表时间：
1989-03-21
期刊：
影响因子：
2.9
通讯作者：
JOB, D
中科院分区：
文献类型：
--
作者：
PATURLE, L;WEHLAND, J;JOB, D
在体外，神经微管蛋白的最大可实现酪氨酸化水平为约50%。我们已经开发了一种方法，以获得一个完整的分离的酪氨酸和nontyrosinatable物种。我们使用免疫亲和柱，偶联YL 1/2单克隆抗体（抗-Tyr-微管蛋白）和快速电泳方法。两个亚群都可以以可聚合的、显然是天然的形式获得。我们发现，大约35%的脑微管蛋白是真正的非酪氨酸，尽管事实上是组装能力。使用针对不可酪氨酸化的微管蛋白的多克隆抗体，我们发现它识别α-微管蛋白上的特异性表位。二聚体的亚基。存在丰富的微管蛋白的亚种，结构上不同的酪氨酸微管蛋白，显然应该记住在免疫荧光研究的分布nontyrosinated微管蛋白在脑组织中。此外，我们还广泛研究了微管蛋白酪氨酸化对微管动力学的影响。尽管比较下的人口的同质性，我们发现没有显着的影响，酪氨酸对微管动力学。同样，微管相关蛋白和STOP蛋白的稳定作用在两个亚群中是相同的。药物紫杉醇似乎更有效地稳定脱酪氨酸微管，但差异是适度的。两者合计，这些发现表明，微管蛋白酪氨酸不影响微管稳定，既不通过修改固有的微管蛋白的性质，也不通过稳定蛋白质的差异结合。最后，完全分离的两种微管蛋白（酪氨酸或去酪氨酸）具有相似的动力学性质，但免疫学不同，应该是在许多动力学研究的微管组装的价值。
The maximum achievable tyrosination level of neurotubulin, in vitro, is about 50%. We have developed a method to obtain a complete separation of the tyrosinatable and nontyrosinatable species. We use an immunoaffinity column, with coupled YL 1/2 monoclonal antibody (anti-Tyr-tubulin) and rapid desalting methods. Both subpopulations can be obtained in a polymerizable, apparently native, form. We find that about 35% of the brain tubulin is truly nontyrosinatable, despite the fact that is assembly competent. Using a polyclonal antibody directed against nontyrosinatable tubulin, we find that it recognizes a specific epitope on the .alpha.-subunit of the dimer. The existence of an abundant tubulin subspecies, structurally different from tyrosinatable tubulin, should obviously be kept in mind in immunofluorescence studies of the distribution of nontyrosinated tubulin in brain tissues. Furthermore, we have extensively investigated the effect of tubulin tyrosination on microtubule dynamics. Despite the homogeneity of the populations under comparison, we find no significant effect of tyrosination on microtubule dynamics. Similarly, the stabilizing effects of microtubule associated proteins and of STOP protein were identical in both subpopulations. The drug taxol seems more efficient in stabilizing detyrosinated microtubules, but the difference is moderate. Taken together, these findings suggest that tubulin tyrosination does not effect microtubule stabilization, neither through modifications of the intrinsic tubulin properties nor through a differential binding of stabilizing proteins. Finally, the complete separation of two tubulin species (tyrosinated or detyrosinated) with similar kinetic properties, but immunologically different, should be of value in many kinetic studies of microtubule assembly.