Turner P F, Margolis R L
J Cell Biol. 1984 Sep;99(3):940-6. doi: 10.1083/jcb.99.3.940.
Taxol has two obvious effects in cells. It stabilizes microtubules and it induces microtubule bundling. We have duplicated the microtubule-bundling effect of taxol in vitro and report preliminary characterization of this bundling using electron microscopy, sedimentation, and electrophoretic analyses. Taxol-bundled microtubules from rat brain crude extracts were seen as massive bundles by electron microscopy. Bundled microtubules sedimented through sucrose five times faster than control microtubules. Electrophoretic analysis of control and taxol-bundled microtubules pelleted through sucrose revealed no striking differences between the two samples except for a protein doublet of approximately 100,000 daltons. Taxol-induced microtubule bundling was not produced by using pure tubulin or recycled microtubule protein; this suggested that taxol-induced microtubule bundling was mediated by a factor present in rat brain crude extracts. Taxol cross-linked rat brain crude extract microtubules were entirely labile to ATP in the millimolar range. This ATP-dependent relaxation was also demonstrated in a more purified system, using taxol-bundled microtubules pelleted through sucrose and gently resuspended. Although the bundling factor did not recycle with microtubule protein, it was apparently retained on isolated taxol-stabilized microtubules. The bundling factor was salt extracted from taxol-stabilized microtubules and its retained activity was demonstrated in an add-back experiment with assembled phosphocellulose-purified tubulin.
紫杉醇在细胞中有两种明显的作用。它能使微管稳定，并诱导微管束的形成。我们已在体外复制了紫杉醇的微管束形成效应，并使用电子显微镜、沉降和电泳分析报告了这种微管束形成的初步特征。通过电子显微镜观察，来自大鼠脑粗提物的紫杉醇束状微管呈现为大量的束。束状微管在蔗糖中的沉降速度比对照微管快五倍。对通过蔗糖沉淀的对照微管和紫杉醇束状微管进行电泳分析，结果显示除了一个约100,000道尔顿的蛋白质双峰外，两个样品之间没有显著差异。使用纯微管蛋白或回收的微管蛋白并不能产生紫杉醇诱导的微管束形成；这表明紫杉醇诱导的微管束形成是由大鼠脑粗提物中存在的一种因子介导的。紫杉醇交联的大鼠脑粗提物微管在毫摩尔范围内对ATP完全不稳定。在一个更纯化的系统中也证实了这种ATP依赖性松弛，该系统使用通过蔗糖沉淀并轻轻重悬的紫杉醇束状微管。尽管束状因子不会与微管蛋白一起循环，但它显然保留在分离的紫杉醇稳定的微管上。束状因子从紫杉醇稳定的微管中用盐提取出来，并且在与组装的磷酸纤维素纯化的微管蛋白进行的回加实验中证明了其保留的活性。
