Dráber P, Dráberová E, Linhartová I, Viklický V
Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague.
Acta Histochem Suppl. 1991;41:9-18.
A panel of monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in microtubules. Limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of tubulin subunits. Antibodies against N-terminal domains were tested for their ability to interfere with the formation of microtubules in vitro. Although the antibodies exhibited similar association constants when tested on immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. The sedimentation assay using microtubules prepared from purified tubulin showed an almost undetectable binding of the antibodies with the strongest inhibitory power to the microtubules. Immunofluorescence staining of unfixed detergent-extracted cells revealed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies against N-terminal domains. The same results were obtained after a microinjection of antibodies into living cells. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of microtubules, considerable regions of the N-terminal domains are not. The surface regions of N-terminal domains appear to be involved in the formation of microtubules.
一组针对α-或β-微管蛋白亚基的单克隆抗体被用于研究微管蛋白分子在微管中的位置。用胰蛋白酶和胰凝乳蛋白酶对微管蛋白进行有限的蛋白水解，然后进行免疫印迹，结果表明这些抗体能够区分微管蛋白亚基的结构域。测试了针对N端结构域的抗体在体外干扰微管形成的能力。尽管这些抗体在固定化微管蛋白上测试时表现出相似的结合常数，但它们对微管组装的抑制作用有所不同。使用从纯化的微管蛋白制备的微管进行的沉降分析表明，具有最强抑制能力的抗体与微管的结合几乎检测不到。对未固定的经去污剂处理的细胞进行免疫荧光染色显示，针对C端结构域决定簇的抗体标记了微管，但这些微管没有被针对N端结构域的抗体修饰。将抗体显微注射到活细胞中后也得到了相同的结果。数据表明，虽然两个亚基的C端结构域部分暴露在微管外部，但N端结构域的相当一部分区域并未暴露。N端结构域的表面区域似乎参与了微管的形成。
