Davis A, Sage C R, Wilson L, Farrell K W
Department of Biological Sciences, University of California, Santa Barbara 93106.
Biochemistry. 1993 Aug 31;32(34):8823-35. doi: 10.1021/bi00085a013.
We describe a method for isolating milligram quantities of assembly-competent tubulin from the budding yeast Saccharomyces cerevisiae. The tubulin is > 95% purified and free of contaminating enzyme activities. As a result, it has been possible to determine the yeast tubulin equilibrium-binding constant for Mg-GTP and the tubulin GTPase activity under nonassembling and assembling conditions. We also determined the critical concentration for yeast tubulin polymerization and found it to be significantly lower than that for bovine brain tubulin under identical conditions. Similarly, the dynamic properties both of individual yeast microtubules and of bulk microtubule suspensions were significantly different from those of bovine brain microtubules free of microtubule-associated proteins. The data suggest that the properties of the yeast tubulin may reflect the particular functional requirements of the yeast cell. With this method, it is now possible to introduce any desired tubulin gene mutation into the budding yeast and correlate the phenotypic effects of the mutation in cells with the effects of the mutation on the biochemical and polymerization properties of the tubulin.
我们描述了一种从出芽酵母酿酒酵母中分离毫克量具有组装能力微管蛋白的方法。该微管蛋白的纯度>95%，且无酶活性污染。因此，得以测定酵母微管蛋白对Mg-GTP的平衡结合常数以及在非组装和组装条件下的微管蛋白GTP酶活性。我们还测定了酵母微管蛋白聚合的临界浓度，发现其在相同条件下显著低于牛脑微管蛋白的临界浓度。同样，单个酵母微管和大量微管悬浮液的动态特性与不含微管相关蛋白的牛脑微管的动态特性显著不同。数据表明，酵母微管蛋白的特性可能反映了酵母细胞的特定功能需求。利用这种方法，现在可以将任何所需的微管蛋白基因突变引入出芽酵母，并将细胞中突变的表型效应与该突变对微管蛋白生化和聚合特性的影响相关联。
