Detrich H W, Wilson L
Biochemistry. 1983 May 10;22(10):2453-62. doi: 10.1021/bi00279a023.
Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.
通过对海胆紫球海胆未受精卵的上清液组分在DEAE - Sephacel或DEAE -纤维素上进行层析，然后在体外进行温度依赖性微管组装和解聚循环，从海胆紫球海胆未受精卵中纯化出微管蛋白。经过两个组装循环后，微管蛋白由α -和β -微管蛋白（占蛋白质的98%以上）以及七种分子量小于55000的微量蛋白质组成；未观察到分子量大于微管蛋白的相关蛋白质。当在尿素 - 聚丙烯酰胺梯度凝胶上进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析时，α -和β -微管蛋白与来自牛脑的对应蛋白并非精确共迁移。二维电泳显示海胆卵微管蛋白包含两种主要的α -微管蛋白和一种主要的β -微管蛋白。在0℃保存的微管蛋白制剂中未观察到寡聚结构。当在含有鸟苷5'-三磷酸的无甘油聚合缓冲液中升温至37℃时，纯化的卵微管蛋白能有效地组装成微管。一次或两次循环的卵微管蛋白组装的临界浓度为0.12 - 0.15mg/mL。通过电子显微镜观察到形态正常的微管，这些微管通过暴露于低温或鬼臼毒素而解聚。在磷酸纤维素上对两次循环的卵微管蛋白制剂进行层析不会改变其蛋白质组成，也不会影响其随后组装成微管。在浓度高于0.5 - 0.6mg/mL时，在组装反应过程中观察到浊度的浓度依赖性“超调”。这些结果表明，卵微管蛋白在没有环状寡聚体和微管相关蛋白的情况下组装成微管，而环状寡聚体和微管相关蛋白是脊椎动物脑微管蛋白的特征。
