MacNeal R K, Purich D L
J Biol Chem. 1978 Jul 10;253(13):4683-7.
A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.
给出了一种制备微管蛋白的方法，即每摩尔6S二聚体含有1摩尔可交换结合的[γ-32P]GTP。研究表明，牛微管蛋白在37℃下能水解添加到正在组装的微管中的每摩尔6S二聚体中的1摩尔GTP。水解和组装以相同的速率且达到相同的程度发生。当去除微管相关蛋白（MAPs）时，水解和组装均无法发生。重新添加MAPs可恢复这两种活性。即使在等摩尔水平下，带有可交换GDP的微管蛋白也会与GTP-微管蛋白共同组装。通过用GDP或GTP进行脉冲追踪实验证明了可交换性。在这些条件下，GDP也是组装的有效抑制剂，在10μM GDP时组装速率降低50%。每摩尔水解的可交换GTP会有1摩尔无机磷酸释放到溶剂中。提出了一种组装机制，其中可交换GTP水解时不存在不可交换GDP的中间转磷酸化过程。
