Studies on the exchangeable nucleotide binding site of tubulin.
Studies on the exchangeable nucleotide binding site of tubulin.
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微管蛋白可交换核苷酸结合位点的研究。
DOI：
10.1111/j.1749-6632.1986.tb38426.x
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发表时间：
1986
影响因子：
5.2
通讯作者：
Himes,RH
中科院分区：
文献类型：
--
作者：
Nath,JP;Eagle,GR;Himes,RH
微管蛋白是不同亚基的二聚体，含有两个鸟嘌呤核苷酸结合位点，其中一个与培养基中的核苷酸交换。为了鉴定包含单个结合位点的亚基，并将微管蛋白的核苷酸结合结构域与其他鸟嘌呤核苷酸结合蛋白进行比较，有必要确定位点周围的一级序列。在这些研究中，通常使用与蛋白质共价相互作用的亲和标记。为了定位和表征可交换的三磷酸鸟苷（GTP）位点，使用了GTP的两种光亲和类似物，8-叠氮基GTP和（3 ′-对叠氮基苯甲酰基）-GTP，以及非光亲和类似物，GTP的高碘酸盐氧化产物，2-（鸟苷甲酰基甲氧基）-3-（三磷酸）丙醛和5 ′-对氟磺酰基苯甲酰基鸟苷（表1）。使用这种类似物的结果导致了不一致的结论。Gaehlen和Haley得出结论，8-叠氮基GTP与0亚基结合，尽管也发生了与a亚基的显著非特异性结合。2最近，Haley等人3报道了β Y亚基被专门标记。Maccioni和Seeds发现3 '-p-叠氮基苯甲酰GTP和2-（鸟苷甲酰甲氧基）-3-（三磷酸）丙醛与α和β亚基的结合同样好，但这种结合是可饱和的，并且与GTP竞争，表明这是由于在可交换的GTP位点上的特异性相互作用。另一方面，Kirsch和Yarbrough的结论是，基于他们发现与GTP没有竞争，用高碘酸盐氧化的GTP标记是非特异性的。使用5 '-对氟磺酰基苯甲酰基鸟苷（一种能够与巯基反应的类似物）也获得了验证数据。在一种情况下：用该化合物处理的微管蛋白能够自组装，并且该化合物被并入p亚基。在另一项研究中，类似物不支持组装，并且不能确定相互作用位点的亚基位置。尽管亲和类似物在确定结合位点方面是有用的，但是在尝试鉴定微管蛋白中可交换核苷酸结合位点处的氨基酸序列时用GTP类似物获得的结果是不明确的。直接光亲和标记具有将天然核苷酸底物或配体共价交联到其结合位点的优点。蛋白质在紫外光的直接作用下与它们的天然配体交联，而无需在任一反应物上引入亲和标记。直接光亲和标记技术已被用于将核苷酸共价连接到许多蛋白质上，以及将核酸共价连接到蛋白质上，但对这些反应的光化学和所形成的加合物的性质知之甚少。它
Tubulin, a dimer of nonidentical subunits, contains two guanine nucleotide binding sites, one of which exchanges with nucleotide in the medium.‘To identify the subunit that contains the individual binding site and to compare the nucleotide binding domain of tubulin with other guanine nucleotide binding proteins, it is necessary to determine the primary sequence around the sites. In such studies use is often made of affinity labels that interact covalently with the protein. To localize and characterize the exchangeable guanosine triphosphate (GTP) site, two photoaffinity analogues of GTP, 8-azido GTP and (3’-p-azido benzoy1)-GTP, as well as nonphotoaffinity analogues, the periodate oxidation product of GTP, 2-(guanylformylmethoxy)-3-(triphospho) propanal and 5’-p-fluorosulfonyl benzoyl guanosine, have been used (TABLE 1). Results from the use of such analogues have led to inconsistent conclusions. Gaehlen and Haley concluded that the 8-azido GTP binds to the 0 subunit, although significant nonspecific binding to the a subunit also occurred. 2 More recently, Haley et af. 3 reported that the (Y subunit was labeled exclusively. Maccioni and Seeds4 found that the 3 ‘-p-azido benzoyl GTP and 2-(guanylformylme-thoxy)-3-(triphospho) propanal bound equally well to the a and p subunits, but the binding was saturable and competitive with GTP, indicating that it was due to a specific interaction at the exchangeable GTP site. On the other hand Kirsch and Yarbrough’concluded that labeling with the periodate oxidized GTP was nonspecific on the basis that they found no competition with GTP. Conflicting data has also been obtained with the use of 5’-p-fluorosulfonyl benzoyl guanosine, an analogue that is capable of reacting with sulfhydryl groups. In one case: tubulin treated with this compound was capable of self-assembly, and the compound was incorporated into the p subunit. In another study’the analogue did not support assembly, and the subunit location of the site of interaction could not be identified. Although affinity analogues are useful in defining binding sites, results obtained with GTP analogues in attempts to identify the amino acid sequence at the exchangeable nucleotide binding site in tubulin have been ambiguous.Direct photoaffinity labeling has the advantage of covalently cross-linking the natural nucleotide substrate or ligand to its binding site in With this method, proteins are cross-linked to their natural ligands under the direct action of ultraviolet light, without the introduction of affinity labels on either of the reactants. The direct photoaffinity labeling technique has been used to covalently link nucleotides to a number of proteins, and nucleic acids to protein^;'^ very little, however is known about the photochemistry of these reactions and the nature of the adducts f~ rmed.’~ It