Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro.
Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro.
复制标题
DOI：
10.1083/jcb.93.3.788
复制
发表时间：
1982-06
期刊：
影响因子：
--
通讯作者：
Rebhun LI
中科院分区：
文献类型：
--
作者：
Keller TC 3rd;Rebhun LI
微管蛋白提取纺锤体分离的海胆的胚胎，在体外重新聚合，并通过三个循环的温度依赖性的组装和拆卸纯化。除微管蛋白外，这些制剂还含有80千道尔顿的蛋白质和少量但可变量的肌动蛋白。在37 ℃下，微管蛋白以0.15-0.2 mg/ml的临界浓度聚合成主要含有14个原丝的光滑壁聚合物。通过DEAE-层析去除80 kdalton蛋白和肌动蛋白不改变聚合的临界浓度。在15摄氏度，这是在生理温度范围内的S。在purpuratus胚胎中，纺锤体微管蛋白将自组装，但总聚合物形成的速率非常慢，需要在试管中数小时。这个速率可以通过剪切聚合微管来增加，从而产生更多的组装末端，这表明聚合物形成的缓慢速率是由于缓慢的自引发速率。如果纺锤体微管蛋白在37摄氏度聚合，然后降低到15摄氏度，一些聚合物将被保留，其百分比取决于蛋白质浓度。这些结果表明，来自S.在不存在可检测的MAP的情况下，紫孢菌将在37 ℃下以低的聚合临界浓度组装，并且将自组装并在生理温度下保持聚合物的稳态水平。
Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus, repolymerized in vitro, and purified through three cycles of temperature-dependent assembly and disassembly. In addition to the tubulin, these preparations contain a protein of 80 kdaltons and a small but variable amount of actin. At 37 degrees C, the tubulin polymerizes with a critical concentration of 0.15-0.2 mg/ml into smooth-walled polymers which contain predominantly 14 protofilaments. Removal of the 80 kdalton protein and the actin by DEAE-chromatography does not change the critical concentration for polymerization. At 15 degrees C, which is within the range of physiological temperatures for S. purpuratus embryos, the spindle tubulin will self-assemble, but the rate of total polymer formation is very slow, requiring hours in the test tube. This rate can be increased by shearing the polymerizing microtubules, creating more ends for assembly, indicating that the slow rate of polymer formation is due to a slow rate of self-initiation. If spindle tubulin is polymerized at 37 degrees C and then lowered to 15 degrees C, some polymer will be retained, the percentage of which depends on the protein concentration. These results demonstrate that spindle tubulin from S. purpuratus will assemble at 37 degrees C with a low critical concentration for polymerization in the absence of detectable MAPs and will self-assemble and maintain steady state levels of polymer at physiological temperatures.