Arévalo M A, Nieto J M, Andreu D, Andreu J M
Centro de Investigaciones Biológicas, C.S.I.C., Madrid, Spain.
J Mol Biol. 1990 Jul 5;214(1):105-20. doi: 10.1016/0022-2836(90)90150-k.
Antibodies to synthetic peptides from the alpha and beta-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences are exposed on the surface of the tubulin alpha beta heterodimer. IgG antibodies to the alpha(430-443) and beta(412-431) sequences perturbed substoichiometrically the assembly of purified tubulin, inducing microtubule bundling and the formation of opened up structures. These positions, which are close to the C termini, were accessible to the anti-peptide antibodies in taxol-induced microtubules, Zn2(+)-induced tubulin sheets, Mg2(+)-induced tubulin rings and in PtK2 cell microtubules. This, together with the comparison of the sizes and gross shapes of the antibody probes and microtubules, suggested that these sequences might be located at the protruding parts of the protofilaments. Antibodies to positions alpha(155-168) did not react with microtubules, while the equivalent zone beta(153-165) was accessible. The alpha(214-226) and beta(241-256) sequences were antigenically occluded in the taxol microtubules, Zn2(+)-induced sheets and Mg2(+)-induced ring arrays, as well as in native microtubules from PtK2 cells, though they became reactive by fixation. This result strongly suggested that these two zones are close to tubulin-tubulin contact sites. A working model is proposed in which the positions alpha(214-226) and beta(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions alpha(241-256) and beta(214-226) are suggested to be related to the alpha-beta binding interface within the heterodimer.
利用针对α和β微管蛋白序列合成肽的抗体，研究该蛋白在微管组装中具有活性的区域。在纯化的小牛脑微管蛋白中，根据其亲水性和保守性选择的六个短序列，在非组装天然条件下进行的竞争放射免疫分析中，被发现可与亲和纯化的免疫球蛋白G（IgG）抗体结合。这表明这六个序列暴露在微管蛋白αβ异二聚体的表面。针对α（430 - 443）和β（412 - 431）序列的IgG抗体以亚化学计量的方式干扰纯化微管蛋白的组装，诱导微管束集并形成开放结构。这些靠近C末端的位置，在紫杉醇诱导的微管、Zn2 +诱导的微管蛋白片层、Mg2 +诱导的微管蛋白环以及PtK2细胞微管中，均可被抗肽抗体识别。这与抗体探针和微管的大小及总体形状的比较一起，表明这些序列可能位于原纤维的突出部分。针对α（155 - 168）位置的抗体不与微管反应，而等效的β（153 - 165）区域是可及的。α（214 - 226）和β（241 - 256）序列在紫杉醇微管、Zn2 +诱导的片层和Mg2 +诱导的环状阵列以及PtK2细胞的天然微管中抗原性被封闭，尽管它们通过固定后变得具有反应性。这一结果强烈表明这两个区域靠近微管蛋白 - 微管蛋白接触位点。提出了一个工作模型，其中α（214 - 226）和β（241 - 256）位置靠近异二聚体之间的轴向接触，这导致原纤维形成，而α（241 - 256）和β（214 - 226）位置被认为与异二聚体内的α - β结合界面有关。
