Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima.
Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima.
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DOI：
10.1083/jcb.98.1.253
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发表时间：
1984-01
期刊：
影响因子：
--
通讯作者：
Rebhun LI
中科院分区：
文献类型：
--
作者：
Suprenant KA;Rebhun LI
具有组装能力的微管蛋白是从未受精和孤雌激活卵母细胞的细胞质中以及从北蛤（Spisula Solidissima）分离的减数分裂纺锤体中纯化出来的。在 22 摄氏度或 37 摄氏度下，Spisula 微管蛋白在第一个聚合周期中组装成 48-51 nm 的大管，在第三个及后续组装周期中组装成 25 nm 的微管。微管由 26-27 个原丝片形成，相对于聚合物长轴以 36 度角螺旋排列，由 α 和 β 微管蛋白以及分子量范围为 30,000 至 270,000 的其他几种蛋白质组成。第三周期微管在横截面中包含14-15个原丝，并且由大于95%的α和β微管蛋白组成。在 37°C 下进行三个聚合循环后，未受精且活化的卵母细胞微管蛋白以 0.09 mg/ml 的临界浓度 (Ccr) 自组装成微管。在22℃的生理温度下，未受精卵母细胞微管蛋白以0.36mg/ml的Ccr组装成微管，以0.42mg/ml的Ccr组装活化的卵母细胞微管蛋白，以0.33mg/ml的Ccr组装分离的减数分裂纺锤体微管蛋白。来自未受精卵母细胞和分离的减数分裂纺锤体的微管蛋白的等电点对于α微管蛋白为5.8，对于β微管蛋白为5.6。此外，卵母细胞和纺锤体α和β微管蛋白的一维肽图即使不相同，也非常相似。这些结果表明，未受精卵母细胞微管蛋白和从第一减数分裂纺锤体分离的微管蛋白在组装特性、等电聚焦和一维肽图谱的基础上无法区分。这些结果表明，微管蛋白从静止卵母细胞状态转变为能够在体内形成纺锤体微管的状态，不需要对微管蛋白进行特殊修饰，但可能涉及微管组织中心或促进组装的微管相关蛋白的可用性的变化。
Assembly-competent tubulin was purified from the cytoplasm of unfertilized and parthogenetically activated oocytes, and from isolated meiotic spindles of the surf clam, Spisula solidissima. At 22 degrees C or 37 degrees C, Spisula tubulin assembled into 48-51-nm macrotubules during the first cycle of polymerization and 25-nm microtubules during the third and subsequent cycles of assembly. Macrotubules were formed from sheets of 26-27 protofilaments helically arranged at a 36 degree angle relative to the long axis of the polymer and were composed of alpha and beta tubulins and several other proteins ranging in molecular weight from 30,000 to 270,000. Third cycle microtubules contained 14-15 protofilaments in cross-section and were composed of greater than 95% alpha and beta tubulins. After three cycles of polymerization at 37 degrees C, unfertilized and activated oocyte tubulin self-assembled into microtubules at a critical concentration (Ccr) of 0.09 mg/ml. At the physiological temperature of 22 degrees C, unfertilized oocyte tubulin assembled into microtubules at a Ccr of 0.36 mg/ml, activated oocyte tubulin assembled at a Ccr of 0.42 mg/ml, and isolated meiotic spindle tubulin assembled at a Ccr of 0.33 mg/ml. The isoelectric points of tubulin from both unfertilized oocytes and isolated meiotic spindles were 5.8 for alpha tubulin and 5.6 for beta tubulin. In addition, one dimensional peptide maps of oocyte and spindle alpha and beta tubulins were very similar, if not identical. These results indicate that unfertilized oocyte tubulin and tubulin isolated from the first meiotic spindle are indistinguishable on the basis of assembly properties, isoelectric focusing, and one dimensional peptide mapping. These results suggest that the transition of tubulin from the quiescent oocyte state to that competent to form spindle microtubules in vivo does not require special modification of tubulin but may involve changes in the availability of microtubule organizing centers or assembly-promoting microtubule-associated proteins.