Williams R C, Detrich H W
Biochemistry. 1979 Jun 12;18(12):2499-503. doi: 10.1021/bi00579a010.
Tubulin can be conveniently separated from the microtubule-associated proteins by chromatography on phosphocellulose [Weingarten, M. D., Lockwood, A. H., Hwo, S.-Y., & Kirschner, M. W. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1858]. Concentrations of Mg2+, GTP, GDP, and glycerol were measured in the various fractions collected during several such separations. The phosphocellulose was found to sequester Mg2+ under the conditions employed for the separation and to retard GTP, GDP, and glycerol relative to tubulin. Polymerization of the resulting phosphocellulose-tubulin was found to be inhibited by this Mg2+ depletion. The composition of the buffer in which tubulin emerges from the chromatographic column was found to depend in a sensitive way upon the retardation of the other components of the buffer and to be a sensitive function of the width of the pooled tubulin peak and of the ratio of the volume of the chromatographic column to that of the column load. The bearing of these findings on interpretation of existing literature is briefly discussed. Efficient separation of tubulin from microtubule-associated proteins can also be obtained on phosphocellulose columns that have been saturated with Mg2+. Such saturation of the column, or addition of Mg2+ to the collected fractions, followed by equilibration of the tubulin with known buffer, is suggested as an improvement to the preparative scheme.
通过磷酸纤维素柱色谱法可方便地将微管蛋白与微管相关蛋白分离[温加滕，M. D.，洛克伍德，A. H.，霍，S.-Y.，& 基尔希纳，M. W.（1975年）《美国国家科学院院刊》72, 1858]。在几次这样的分离过程中收集的各个组分中测量了Mg2+、GTP、GDP和甘油的浓度。发现在用于分离的条件下，磷酸纤维素会螯合Mg2+，并且相对于微管蛋白会阻滞GTP、GDP和甘油。发现这种Mg2+耗竭会抑制所得磷酸纤维素 - 微管蛋白的聚合。发现微管蛋白从色谱柱中流出时缓冲液的组成敏感地取决于缓冲液其他成分的阻滞情况，并且是合并的微管蛋白峰宽度以及色谱柱体积与柱负载体积之比的敏感函数。简要讨论了这些发现对解释现有文献的意义。在已用Mg2+饱和的磷酸纤维素柱上也可有效地将微管蛋白与微管相关蛋白分离。建议对制备方案进行改进，即对柱子进行这种饱和处理，或向收集的组分中添加Mg2+，然后用已知缓冲液使微管蛋白平衡。
