Paturle L, Wehland J, Margolis R L, Job D
Laboratoire BRCE, INSERUM U 244, Centre d'Etudes Nucléaires, Grenoble, France.
Biochemistry. 1989 Mar 21;28(6):2698-704. doi: 10.1021/bi00432a050.
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 it 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.
在体外，神经微管蛋白可达到的最大酪氨酸化水平约为50%。我们开发了一种方法来完全分离可酪氨酸化和不可酪氨酸化的物种。我们使用了一个偶联有YL 1/2单克隆抗体（抗酪氨酸化微管蛋白）的免疫亲和柱和快速脱盐方法。两种亚群都可以以可聚合的、明显天然的形式获得。我们发现，尽管脑微管蛋白具有组装能力，但约35%的脑微管蛋白是真正不可酪氨酸化的。使用针对不可酪氨酸化微管蛋白的多克隆抗体，我们发现它识别二聚体α亚基上的一个特定表位。在对脑组织中未酪氨酸化微管蛋白分布的免疫荧光研究中，显然应牢记存在一种结构上不同于可酪氨酸化微管蛋白的丰富微管蛋白亚群。此外，我们广泛研究了微管蛋白酪氨酸化对微管动力学的影响。尽管所比较的群体具有同质性，但我们发现酪氨酸化对微管动力学没有显著影响。同样，微管相关蛋白和STOP蛋白在两个亚群中的稳定作用是相同的。药物紫杉醇似乎在稳定去酪氨酸化微管方面更有效，但差异不大。综上所述，这些发现表明，微管蛋白酪氨酸化既不通过改变微管蛋白的内在特性，也不通过稳定蛋白的差异结合来影响微管的稳定。最后，两种具有相似动力学特性但免疫特性不同的微管蛋白物种（酪氨酸化或去酪氨酸化）的完全分离，在微管组装的许多动力学研究中应该是有价值的。
