GTP analogues interact with the tubulin exchangeable site during assembly and upon binding.
GTP analogues interact with the tubulin exchangeable site during assembly and upon binding.
复制标题
GTP 类似物在组装过程中和结合后与微管蛋白可交换位点相互作用。
DOI：
10.1021/bi00457a017
复制
发表时间：
1990
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：
Mejillano,MR;Barton,JS;Nath,JP;Himes,RH
1989年9月25日收到的摘要：非水解核苷酸类似物和其他核苷三磷酸是否支持微管蛋白组装的问题得到了解决。在可交换位点含有残留GTP的微管蛋白，在DMSO或甘油存在下，在不加入GTP的情况下聚合。在达到最大吸光度后，以缓慢的速率发生分解。当0.5 mM GMPPCP、GMPPNP或ATP包括在装配反应中时，不发生拆卸，并且每摩尔微管蛋白约0.1 mol这些核苷酸掺入蛋白质中。当使用5 mM核苷酸或在不可水解类似物的情况下包括碱性磷酸酶时，发生更大量的组装，并掺入约0.7- 0.8mol类似物。组装反应的产物是冷不稳定的微管和原丝带。在微管和带的冷解聚之后，第二次组装循环产生一些微管，但冷稳定的无定形聚合物是主要产物。此外，当GTP在可交换的网站是第一次通过一个循环的组装，然后解聚，在GMPPCP，GMPPNP，或ATP的存在下组装产生的微管和冷稳定的聚合物的混合物，这两者都包含绑定类似物。GMPPCP、GMPPNP或ATP掺入聚合的微管蛋白总是以牺牲可交换位点的GDP为代价，其含量相应减少。在非组装条件下，用5 mM GMPPCP、GMPPNP或ATP孵育微管蛋白也取代GDP。GMPPCP的结合是在从可交换位点产生GDP和GTP的最大去除的条件下测量的，并且获得（5.3-7.1）X 104 M-1的a。对其他核苷酸的研究表明，在5 mM浓度的ITP、UTP和CTP的存在下，组装导致微管和条带在达到稳态后不解聚。在ITP的情况下，在聚合蛋白中发现IDP，但没有证据表明UTP、CTP、UDP或CDP的掺入。嘧啶核苷酸的稳定作用是由于微管蛋白制剂中存在低水平的核苷二磷酸激酶活性。从这些研究中，我们得出结论，几个嘌呤核苷三磷酸可以结合到可交换的核苷酸位点，在微管种子的存在下，含有这些核苷酸的微管蛋白参与聚合的延伸阶段。在没有微管种子的情况下，在所用条件下的聚合导致一些微管，但主要产物是微管蛋白的无定形冷稳定聚合物。因此，在可交换位点具有类似物的微管蛋白似乎具有降低的参与微管聚合过程的成核阶段的能力。
Revised Manuscript Received September 25, 1989 abstract: The question of whether nonhydrolyzable nucleotide analogues and other nucleoside triphosphates support tubulin assembly was addressed. Tubulin which contained residual GTP at the exchangeable site polymerized in the absence of added GTP inthe presence of DMSO or glycerol. After maximum absorbance was reached, disassembly occurred at a slow rate. When 0.5 mM GMPPCP, GMPPNP, or ATP was included in the assembly reaction, disassembly did not occur, and about 0.1 mol of these nucleotides per mole of tubulin was incorporated into the protein. When 5 mM nucleotide was used or alkaline phosphatase was included in the case of the nonhydrolyzable analogues, a greater amount of assembly occurred and about 0.7-0.8 mol of analogue was incorporated. The products of the assembly reaction were cold-labile mi-crotubules and protofilament ribbons. After cold-depolymerization of the microtubules and ribbons, a second cycle of assembly produced some microtubules, but cold-stable amorphous polymers were the major product. In addition, when GTP at the exchangeable site was first removed by a cycle of assembly, followed by depolymerization, assembly in the presence of GMPPCP, GMPPNP, or ATP produced a mixture of mi-crotubules and cold-stable polymers, both of which contained bound analogue. Incorporation of GMPPCP, GMPPNP, or ATP into polymerized tubulin always occurred atthe expense of GDP at the exchangeable site, the content of which decreased correspondingly. Incubation of tubulin with 5 mM GMPPCP, GMPPNP, or ATP under nonassembly conditions also displaced GDP. Binding of GMPPCP was measured under conditions which produced the maximum removal of GDP and GTP from the exchangeable site, and a of (5.3-7.1) x 104 M'1 was obtained. Studies with other nucleotides showed that assembly in the presence of 5 mM concentrations of ITP, UTP, and CTP led to microtubules and ribbons which did not depolymerize after reachingsteady state. In the case of ITP, IDP was found in the polymerized protein, but no evidence for the incorporation of UTP, CTP, UDP, or CDP was obtained. The stabilization by the pyrimidine nucleotides was due to a low level of nucleoside-diphosphate kinase activity present in the tubulin preparation. From these studies, we conclude that several purine nucleoside triphosphates can bind to the exchangeable nucleotide site and, in the presence of microtubule seeds, tubulin containing these nucleotides participates in the elongation phase of polymerization. In the absence of microtubule seeds, polymerization under the conditions used leads to some microtubules, but the major product is an amorphous cold-stable polymer of tubulin. Tubulin with analogues at the exchangeable site, therefore, appears to have a reduced ability to participate in the nucleation phase of the microtubule polymerization process.