Mechanism of assembly of sea urchin egg tubulin.
Mechanism of assembly of sea urchin egg tubulin.
复制标题
海胆卵微管蛋白的组装机制。
DOI：
10.1111/j.1749-6632.1986.tb38430.x
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发表时间：
1986
影响因子：
5.2
通讯作者：
Jordan,MA
中科院分区：
文献类型：
--
作者：
Detrich3rd,HW;Jordan,MA
从海胆 Strongylocentrotus purpuratus 卵中纯化的微管蛋白可在生理（18 摄氏度）和非生理（37 摄氏度）温度下在体外有效组装形成微管。组装反应不需要 MAP、环状低聚物和高浓度的非生理溶剂。当浓度高于 1.2 mg/ml（18 摄氏度）和 0.5 mg/ml（37 摄氏度）时，观察到浊度和小角度光散射的浓度依赖性超调：浊度和散射迅速增加至峰值，然后渐近下降至稳态值。电子显微镜分析表明，微管蛋白片在超调组装的初始阶段普遍存在，而完整的微管在稳定状态下存在。溶液双折射的定性观察表明，聚合物在组装过程中逐渐变得更加排列。超调不能用微管蛋白的蛋白水解或变性、GTP 的消耗、组装质量的减少或聚合物长度的重新分布来解释。综上所述，结果表明，组装聚合物的形式和/或组织的变化是导致浊度和小角度光散射超调的原因。我们的结果与微管组装模型一致，该模型假设微管蛋白片成核并随后折叠片以形成成熟的微管。
Tubulin purified from eggs of the sea urchin Strongylocentrotus purpuratus assembles efficiently in vitro to form microtubules at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. MAPs, ring oligomers, and high concentrations of nonphysiological solvents are not required for the assembly reaction. At concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C a concentration-dependent overshoot in turbidity and in light scattering at small angles was observed: turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Electron microscopic analysis demonstrated that tubulin sheets were prevalent during the initial stages of overshoot assembly, whereas complete microtubules were present at steady state. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. The overshoot cannot be explained by proteolysis or denaturation of tubulin, by depletion of GTP, by a decrease in assembled mass, or by redistribution of polymer lengths. Taken together, the results suggest that changes in the form and/or in the organization of the assembling polymer are responsible for the overshoots in turbidity and in light scattering at small angles. Our results are consistent with models of microtubule assembly that postulate nucleation by tubulin sheets and subsequent folding of the sheets to form mature microtubules.