Detraves C, Mazarguil H, Lajoie-Mazenc I, Julian M, Raynaud-Messina B, Wright M
C.N.R.S., Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.
Cell Motil Cytoskeleton. 1997;36(2):179-89. doi: 10.1002/(SICI)1097-0169(1997)36:2<179::AID-CM7>3.0.CO;2-4.
The presence of gamma-tubulin in microtubule preparations, obtained by disassembly/ assembly cycles at 0degreesC/37degreesC from the brain of several mammals, is demonstrated by immunoblotting with specific antibodies directed against three distinct regions of the protein. In contrast gamma-tubulin was absent from pure tubulin obtained by chromatography on phosphocellulose, but was retained on the column with the other microtubule-associated proteins. A large part of the gamma-tubulin was present in cold stable material remaining after microtubule disassembly at OdegreesC and was partially solubilized using high salt, thus preventing its purification by the usual assembly/disassembly procedure used for alpha/beta-tubulin heterodimers. Brain gamma-tubulin was purified by affinity chromatography with gamma-tubulin antibodies raised against its carboxyl terminal region. Purified gamma-tubulin consisted of at least two polypeptides present in equal quantities and exhibiting a pI of 6.5 and 6.6, respectively. It was associated with the alpha/beta-tubulin heterodimer and with at least five other polypeptides of 75, 105, 130, 195, and 250 kDa. With the exception of the 250 kDa polypeptide, all of these proteins seem to be present in gamma-tubulin complexes isolated from Xenopus eggs. But, in contrast with Xenopus egg complexes, brain complexes exhibited a considerable heterogeneity of their apparent masses and composition in sucrose gradient centrifugation, in agreement with the absence of an homogeneous structure in electron microscopy. Despite this heterogeneity, gamma-tubulin complexes bind quantitatively to microtubule extremities. The possibility to further use mammalian brain gamma-tubulin and some of its associated proteins in biochemical and pharmacological experiments is of interest since brain microtubule protein preparations have been extensively used for studying both microtubule dynamics and the activity of microtubule poisons.
通过在0℃/37℃下对几种哺乳动物大脑进行拆卸/组装循环获得的微管制剂中γ-微管蛋白的存在，通过用针对该蛋白质三个不同区域的特异性抗体进行免疫印迹来证明。相比之下，通过磷酸纤维素柱层析获得的纯微管蛋白中不存在γ-微管蛋白，但它与其他微管相关蛋白一起保留在柱上。大部分γ-微管蛋白存在于0℃下微管拆卸后剩余的冷稳定物质中，并使用高盐部分溶解，因此无法通过用于α/β-微管蛋白异二聚体的常规组装/拆卸程序进行纯化。通过用针对其羧基末端区域产生的γ-微管蛋白抗体进行亲和层析，纯化了脑γ-微管蛋白。纯化的γ-微管蛋白由至少两种等量存在的多肽组成，其pI分别为6.5和6.6。它与α/β-微管蛋白异二聚体以及至少五种其他75、105、130、195和250 kDa的多肽相关。除了250 kDa的多肽外，所有这些蛋白质似乎都存在于从非洲爪蟾卵中分离的γ-微管蛋白复合物中。但是，与非洲爪蟾卵复合物不同，脑复合物在蔗糖梯度离心中表现出明显的质量和组成的相当大的异质性，这与电子显微镜中不存在均匀结构一致。尽管存在这种异质性，γ-微管蛋白复合物仍定量结合到微管末端。由于脑微管蛋白制剂已被广泛用于研究微管动力学和微管毒物的活性，因此在生化和药理学实验中进一步使用哺乳动物脑γ-微管蛋白及其一些相关蛋白的可能性很有意义。
