Bayley P M, Butler F M, Clark D C, Manser E J, Martin S R
Biochem J. 1985 Apr 15;227(2):439-55. doi: 10.1042/bj2270439.
The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.
在一系列pH值、温度、核苷酸和蛋白质浓度条件下，对牛和猪的微管蛋白在体外组装的动力学进行了研究。动力学通常是双相的，有两个主要过程，幅度相似，但速率相差一个数量级。速率和幅度是溶液条件的复杂函数。快相和慢相的速率随着蛋白质浓度的增加而达到极限值，并且在pH 6.5时比pH 6.95时受到更严格的限制。这种行为表明，在较高蛋白质浓度下，除了微管蛋白二聚体的缩合聚合机制之外的其他机制成为限速因素。光散射的波长依赖性和超微结构标准的恒定性表明，在组装过程的两个阶段都形成了正常形态的微管。对正在组装的微管蛋白进行电泳分析表明，在快相中形成了富含微管相关蛋白（MAP）的微管。微管蛋白稀释时寡聚体的解离速率在大小和温度依赖性上与快相速率密切平行。我们提出，这个过程的速率构成了组装快相速率的上限。MAP从富含MAP的微管中重新分布的动力学可能是限制慢相速率的一个因素。推导了一个微管蛋白自组装的工作模型，该模型纳入了解离和重新分布机制，这些机制对双分子加成反应可达到的组装速率施加了上限。在微管伸长的两个阶段，含MAP的片段都起着关键作用。推断动力学行为随溶液条件的变化源于快速温度跃升后寡聚体形成的片段的性质和特性。该模型解释了极限速率行为，并指出了用于评估替代途径相对贡献的实验标准。
