Activation of the dynein adenosinetriphosphatase by microtubules.
Activation of the dynein adenosinetriphosphatase by microtubules.
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微管激活动力蛋白腺苷三磷酸酶。
DOI：
10.1021/bi00350a022
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Johnson,KA
中科院分区：
文献类型：
--
作者：
Omoto,CK;Johnson,KA
宾夕法尼亚州立大学分子和细胞生物学系，大学公园，宾夕法尼亚州16802接收于1985年6月3日;修订的Mandarin pt接收于1985年9月12日摘要：先前的工作已经表明，在微管-动力蛋白复合物的快速三磷酸腺苷（ATP）诱导的解离之后，ATP酶中的限速步骤是产物释放[约翰逊，KA（1983）J.Biol.Chem.258，13825-13832]，其以约2- 6s-1的速率发生。在本报告中，我们通过检查微管对产物释放速率的影响来完成对ATP酶循环的分析。在这些研究中，我们使用了重新聚合的四膜虫轴丝微管和无微管相关蛋白（MAP）的牛脑微管，这些微管被证明没有任何可测量的ATP酶活性。四膜虫22 S动力蛋白主要通过ATP敏感位点与这些微管结合，其结合速率为（0.2-1）× 106 M~（-1），比含MAP的脑微管的结合速率高50倍。ATP诱导的微管动力蛋白复合物的快速解离的表观二阶速率常数与ATP浓度等于1.6 × 106 M_1 s_1，这个值仅略低于在MAP的存在下观察到的。在ATP诱导的解离后，由于水解ATP所需的时间，动力蛋白在滞后期后与微管重新结合。随着微管浓度的增加，再结合的滞后时间缩短，表明微管增加了ATP周转率。在稳态下的直接测量结果表明，比活性的动力蛋白的微管浓度增加。这些数据为动力蛋白ATP酶在溶液中被微管激活提供了明确的证据，在该机制中，动力蛋白产物中间体与微管的重新结合[以1.5的速率]。（1.2-6）X 104* M-1 s-1]提高产物释放速率。大量工作已经根据方案I建立了微管-动力蛋白ATP酶的前两个步骤，其中M代表微管，D代表动力蛋白。腺苷5 '-三磷酸（ATP）1的结合（步骤1）诱导动力蛋白从微管中快速解离（步骤2），随后是较慢的水解反应（步骤3）
Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 Received June 3, 1985; Revised Manuscript Received September 12, 1985 abstract: Previous work has indicated that following the rapid adenosine 5'-triphosphate (ATP) induced dissociation of the microtubule-dynein complex, the rate-limiting step in the ATPasecycle is product release [Johnson, KA (1983) J. Biol. Chem. 258, 13825-13832], which occurs at a rate of approximately 2-6 s" 1. In this report we complete the analysis of the ATPase cycle by examining the effect of microtubules on the rate of product release. For these studies we used repolymerized Tetrahymena axonemal microtubules and microtubule-associatedprotein (MAP) free bovine brain microtubules which were shown to be free of any measurable ATPase activity. Tetrahymena 22S dynein bound to these microtubules predominantly by the ATP-sensitive site and at a rate giving an apparent second-order rate constant of (0.2-1) X 106 M~'s’1, which is 50-fold greater than the rate observed with brainmicrotubules containing MAPs. ATP induced the rapid dissociation of the microtubule-dynein complex with an apparent second-order rate constant vs. ATP concentration equal to 1.6 X 106 M_1 s_1; this value is only slightly lower than that observed in the presence of MAPs. After the ATP-induced dissociation, the dynein reassociated with the microtubules following a lag period due to the time required to hydrolyze the ATP. The duration of the lag timefor reassociation decreased with increasing microtubule concentration, suggesting that microtubules increased the rate of ATP turnover. Direct measurements at steady state showed that the specific activity of the dynein increased with increasing microtubule concentration. These data provide clear evidence for activation of the dynein ATPase by microtubules in solution by a mechanism in which the rebinding of the dynein-product intermediate to the microtubules [at a rate of (1.2-6) X 104* M" 1 s_1] enhances the rate of product release.I^ evious work has established the first two steps of the microtubule-dynein ATPase according to Scheme I, where M represents a microtubule and D represents dynein. The binding of adenosine 5'-triphosphate (ATP) 1 (step 1) induces a rapid dissociation of dynein from the microtubule (step2) which is followed by a slower hydrolysis reaction (step 3)