Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate.
Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate.
复制标题
用 5-三磷酸鸟苷直接光亲和标记微管蛋白。
DOI：
10.1021/bi00327a040
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Himes,RH
中科院分区：
文献类型：
--
作者：
Nath,JP;Eagle,GR;Himes,RH
在254 nm处，微管蛋白在[~3H]GTP或[~H]GDP存在下照射，导致核苷酸共价掺入蛋白质中。标记的特异性表现为：随着[~H]GTP浓度的增加，标记量增加到平台值，达到饱和的比例约为1.5；GTP/微管蛋白比值为1和100时，获得相同的标记量；[~H]GMP不掺入二聚体，也不抑制[~H]GTP的掺入；[~H]ATP不掺入；[~H]GTP不掺入变性的微管蛋白或血清白蛋白中。当用[a-32P]GTP进行辐照实验时，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法显示，所结合的标记与8亚基结合。辐射处理确实引起微管蛋白分子的变化，导致组装能力和巯基的下降，但当辐射过程中存在大量过剩的GTP时，这些影响最小。在组装状态下微管蛋白的标记比在自由态下观察到的要少得多。微管蛋白的主要成分1微管蛋白是微管蛋白100000的二聚体。该二聚体由不完全相同的单体a和8组成，含有2摩尔的鸟嘌呤核苷酸，一个位于易交换的位置，另一个位于不可交换的位置(Weisenberg等人，1968)。当微管蛋白聚合形成MTS时，可交换的核苷酸被水解成GDP(Kobayashi，1975；Weisenberg等人，1976)，尽管在某些条件下，水解与组装不一致(Carlier&Pantaloni，1981)。
Irradiation of tubulin in the presence of [3H] GTP or [3H] GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H] GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100;[3H] GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H] GTP;[3H] ATP was not incorporated;[3H] GTP incorporation did not occur into denatured tubulin or into serum albumin. When [a-32P] GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the 8 subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin inthe assembled state was much less than that observed in the freestate. e principal proteincomponent of microtubules (MTs), 1 tubulin, is a dimer of MT100000. The dimer, which consists of nonidentical monomers, a and 8, contains 2 mol of guanine nucleotide, one at a readily exchangeablesite and the other at a nonexchangeable site (Weisenberg et al., 1968). Upon polymerization of tubulin to form MTs, the exchangeable nucleotide is hydrolyzed to GDP (Kobayashi, 1975; Weisenberg et al., 1976), although under some conditions hydrolysis is not coincident with assembly (Carlier & Pantaloni, 1981).