LOCALIZATION OF THE COLCHICINE-BINDING SITE OF TUBULIN
LOCALIZATION OF THE COLCHICINE-BINDING SITE OF TUBULIN
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DOI：
10.1073/pnas.90.24.11598
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发表时间：
1993-12-15
影响因子：
11.1
通讯作者：
WOLFF, J
中科院分区：
文献类型：
--
作者：
UPPULURI, S;KNIPLING, L;WOLFF, J
我们之前已经证明，大鼠脑微管蛋白是一种由 α 和 β 单体组成的异二聚体，可以通过近紫外线照射用 [H-3] 秋水仙碱共价标记。大多数标签出现在β-微管蛋白中。我们在此表明，β-微管蛋白可以从 SDS 制备凝胶中分离和纯化，并通过蛋白水解进行分析。胰凝乳蛋白酶产生一条几乎等于 4 kDa 的标记条带，其中包含两个肽。胰蛋白酶消化还产生了一条几乎等于 4 kDa 的条带，其中包含两个肽。序列分析揭示了胰凝乳蛋白酶的残基1-36和213-242的肽以及胰蛋白酶的残基1-46和214-241的肽。为了确定哪种肽带有标记，用胰蛋白酶对 β-微管蛋白进行有限水解；该过程产生了经抗体鉴定的标记的 16-kDa N 端肽和 35-kDa C 端肽。分离这些肽并用胰蛋白酶进行广泛消化，产生两个标记的肽，对应于 16-kDa N 端片段的残基 1-46 和 35-kDa C 端片段的残基 214-241。这些结果表明，β-微管蛋白中的至少两个区域特异性参与秋水仙碱结合，并且秋水仙碱分子的跨度小于或等于11埃，桥接天然β单体中的这两个区域。
We have previously shown that rat brain tubulin, a heterodimer consisting of an alpha and beta monomer, can be covalently labeled with [H-3]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 almost-equal-to 4-kDa band that contained two peptides. Tryptic digestion also yielded an almost-equal-to 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, less-than-or-equal-to 11 angstrom, bridges these two regions in the native beta monomer.