A quantitative analysis of microtubule elongation.
A quantitative analysis of microtubule elongation.
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DOI：
10.1083/jcb.71.3.749
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发表时间：
1976-12
期刊：
影响因子：
--
通讯作者：
Bryan J
中科院分区：
文献类型：
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作者：
Bryan J
已开发出差别化抑制体外微管成核和延长的方法。通过使用聚阴离子，可以制备几毫克/毫升的具有组装能力的微管蛋白溶液，并且在实验过程中不会表现出明显的自发组装。微管延长可以通过添加已知数量的微管片段来启动。对结果过程的详细分析表明：(A)环不是成核序列中的必需中间体，环和原丝片都不是延伸反应中的必需中间体。(B)细长的微管末端通常有一小段开放的原细丝片或“C-微管”，类似于在体内观察到的。(C)浊度的发展遵循接近平衡值的简单指数方法。(D)最终平衡值与添加成核碎片的数量无关，而达到平衡的初始增长速度和半时间取决于添加的原子核数量。(E)平衡状态下微管的最终长度与添加的碎片数量成反比。(F)平衡常数与微管长度无关。(G)每个微管的组装和拆卸位置的数量不是微管长度的函数。(H)微管的正向速率常数、最终聚合物浓度和生长速率取决于存在的聚阴离子浓度。这些结果有力地支持了微管组装是“缩聚-聚合”的观点，并提供了关于体外伸长的动力学和长度分布的基本信息。
Methods have been developed for differentially inhibiting microtubule nucleation and elongation in vitro. By use of polyanions, assembly- competent tubulin solutions of several milligrams/milliliter can be prepared which do not exhibit appreciable spontaneous assembly during the time-course of an experiment. Microtubule elongation can be initiated by the addition of known numbers of microtubule fragments. A detailed analysis of the resulting process demonstrates that: (a) rings are not obligatory intermediates in the nucleation sequence, and neither rings nor protofilament sheets are obligatory intermediates in the elongation reaction. (b) The end of an elongating microtubule often has a short region of open protofilament sheet or "C-microtubule" similar to that observed in vivo. (c) The development of turbidity follows a simple exponential approach to an equilibrium value. (d) The final equilibrium values are independent of the number of added nucleating fragments, while the initial growth rates and half-times to reach equilibrium are dependent on the number of added nuclei. (e) The final lengths of the microtubules at equilibrium are inversely proportional to the number of added fragments. (f) The equilibrium constants are independent of microtubule length. (g) The number of assembly and disassembly sites per microtubule is not a function of microtubule length. (h) The forward rate constants, the final polymer concentrations, and growth rates of microtubules are dependent upon the concentration of polyanion present. These results are strongly supportive of the idea that microtubule assembly is a "condensation- polymerization" and provide basic information on the kinetics and length distributions of the elongation in vitro.