de la Viña S, Andreu D, Medrano F J, Nieto J M, Andreu J M
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Biochemistry. 1988 Jul 12;27(14):5352-65. doi: 10.1021/bi00414a060.
We synthesized five peptides homologous to the potentially antigenic positions alpha(214-226), alpha(430-443), alpha(415-443), beta(241-256), and beta(412-431) of the porcine brain tubulin sequences. These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions alpha(415-430) and beta(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones alpha(415-430) and beta(412-431). C-Terminal cleavages by protease V8 are preferential of beta-tubulin. All six proteases tested cleave alpha- and/or beta-tubulin at one or both of the internal zones. These zones are located roughly at one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.
我们合成了与猪脑微管蛋白序列中潜在抗原位置α(214 - 226)、α(430 - 443)、α(415 - 443)、β(241 - 256)和β(412 - 431)同源的五种肽。这些肽成功地用于产生微管蛋白交叉反应抗体。这些抗体对肽所跨越的微管蛋白区域具有特异性。它们在免疫印迹中与微管蛋白条带特异性反应，在细胞骨架的免疫荧光测定中与微管特异性反应。C末端区域的肽也已用于分别定位两种现有的针对微管蛋白的单克隆抗体在α(415 - 430)和β(412 - 431)位置识别的决定簇。在抗肽抗体的首次应用中，我们绘制了纯化的小牛脑微管蛋白经胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶、嗜热菌蛋白酶、枯草杆菌蛋白酶和金黄色葡萄球菌蛋白酶V8进行有限蛋白水解的片段。已鉴定出37种肽，其中32种基于其抗原反应性已明确排列到微管蛋白序列中。蛋白酶有三个主要的、明确界定的优先切割区域：C末端和每条链中的两个内部区域。枯草杆菌蛋白酶对两条链的C末端切割不会消除α(415 - 430)和β(412 - 431)区域的抗原反应性。蛋白酶V8的C末端切割优先作用于β-微管蛋白。所测试的所有六种蛋白酶在一个或两个内部区域切割α-和/或β-微管蛋白。这些区域大致位于两个亚基链长度的三分之一和三分之二处。因此，提出了一个微管蛋白单体模型，该模型由三个主要的、由蛋白水解定义的紧密区域（N末端、中间和C末端三分之一）和可切割区域组成。结合目前可用的微管蛋白结构信息对该模型进行了讨论。
