Localization of the colchicine-binding site of tubulin.

We have previously shown that rat brain tubulin, a heterodimer consisting of an alpha and beta monomer, can be covalently labeled with [3H]colchicine by near UV irradiation. Most of the label appears in beta-tubulin. We show here that beta-tubulin can be separated and purified from SDS preparative gels and analyzed by proteolysis. Chymotrypsin yielded a labeled approximately 4-kDa band that contained two peptides. Tryptic digestion also yielded an approximately 4-kDa band containing two peptides. Sequence analysis revealed a peptide of residues 1-36 and 213-242 for chymotrypsin and a peptide of residues 1-46 and 214-241 for trypsin. To identify which peptide carried the label, limited hydrolysis of beta-tubulin was done with trypsin; this procedure yielded a labeled 16-kDa N-terminal peptide and a 35-kDa C-terminal peptide, as identified by antibodies. Isolation of these peptides and extensive digestion with trypsin yielded two labeled peptides corresponding to residues 1-46 from the 16-kDa N-terminal fragment and residues 214-241 from the 35-kDa C-terminal fragment. These results show that at least two regions in beta-tubulin are specifically involved in colchicine binding and that the span of the colchicine molecule, < or = 11 A, bridges these two regions in the native beta monomer. IMAGES:

此前我们已证实，大鼠脑微管蛋白（tubulin）——一种由α和β亚基单体构成的异二聚体——可通过近紫外照射与[3H]秋水仙碱完成共价标记，且绝大多数标记信号分布于β微管蛋白中。本文进一步证明，可从SDS制备型凝胶中分离并纯化β微管蛋白，随后开展蛋白水解分析。胰凝乳蛋白酶水解可得到一条约4kDa的标记条带，该条带包含两段肽段；胰蛋白酶水解同样可获得一条约4kDa的标记条带，其中亦包含两段肽段。序列分析结果显示，胰凝乳蛋白酶酶解得到的肽段对应氨基酸残基1-36与213-242，而胰蛋白酶酶解得到的肽段对应残基1-46与214-241。为明确携带标记物的肽段，我们使用胰蛋白酶对β微管蛋白进行有限水解，通过抗体鉴定得到一条16kDa的N端标记肽段，以及一条35kDa的C端肽段。对上述两段肽段进行分离并经胰蛋白酶深度酶解后，得到两段标记肽段，分别对应16kDa N端片段的1-46位残基，以及35kDa C端片段的214-241位残基。上述结果表明，β微管蛋白中至少存在两个区域特异性参与秋水仙碱结合，且秋水仙碱分子的跨度≤11埃，可桥接天然β单体中的这两个区域。图片：