Suprenant K A, Rebhun L I
J Biol Chem. 1983 Apr 10;258(7):4518-25.
Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by DEAE-column chromatography and cycles of temperature-dependent assembly and disassembly. Tubulin-containing column fractions self-assemble into intact microtubules in the absence of high molecular weight microtubule-associated proteins. Egg microtubules assembled during the third cycle of assembly following DEAE-chromatography are composed of 2 or 3 alpha tubulins and 2 beta tubulins as assayed by isoelectric focusing and two-dimensional electrophoresis. The critical protein concentrations necessary for assembly of egg tubulin at 37 and 25 degrees C are 0.15-0.24 and 0.24-0.28 mg/ml, respectively. At physiological temperatures, the critical protein concentrations are 0.81 mg/ml at 15 degrees C and 0.70-0.79 mg/ml at 18 degrees C. At 18 degrees C, bovine brain microtubule-associated proteins stoichiometrically stimulate the initial rate and final extent of egg tubulin assembly. These hybrid microtubules assemble at 18 degrees C at a critical protein concentration of 4-20 micrograms/ml.
通过DEAE柱色谱法以及温度依赖性组装和解聚循环，从海胆紫球海胆未受精的卵中纯化出微管蛋白。在没有高分子量微管相关蛋白的情况下，含有微管蛋白的柱级分可自组装成完整的微管。经DEAE色谱后，在组装的第三个循环中组装的卵微管，通过等电聚焦和二维电泳分析，由2或3个α微管蛋白和2个β微管蛋白组成。在37℃和25℃下，卵微管蛋白组装所需的临界蛋白浓度分别为0.15 - 0.24mg/ml和0.24 - 0.28mg/ml。在生理温度下，15℃时的临界蛋白浓度为0.81mg/ml，18℃时为0.70 - 0.79mg/ml。在18℃时，牛脑微管相关蛋白以化学计量方式刺激卵微管蛋白组装的初始速率和最终程度。这些杂交微管在18℃下以4 - 20μg/ml的临界蛋白浓度组装。
