Spiegelman B M, Penningroth S M, Kirschner M W
Cell. 1977 Nov;12(3):587-600. doi: 10.1016/0092-8674(77)90259-8.
Radioactively labeled tubulin from Chinese hamster ovary (CHO) cells can be isolated by co-polymerization with nonradioactive porcine brain microtubule protein. 75% of the soluble tubulin in CHO extracts co-polymerizes with the porcine protein through several cycles, without preferential loss of either CHO or porcine subunits. After phosphocellulose chromatography of the co-polymerized microtubules, the CHO tubulin is radiochemically homogeneous, as judged by SDS-polyacrylamide gel electrophoresis. CHO tubulin purified in this way has 1 mole of nucleotide per mole of protein noncovalently bound at the non-exchangeable or N site. This-layer chromatography indicates that the N site nucleotide is entirely ribo-GTP. Label and chase experiments show that the N site GTP exchanges intracellularly with a half-time of 33 hr in growing cells which have a generation time of 17 hr, while the tubulin polypeptides are degraded with a half-time of 48 hr. Intracellular hydrolysis of the gamma-phosphate of the N site nucleotide can be detected but occurs very slowly, with a half-time of 24 hr. These results suggest that the N site nucleotide may function in vivo as a stable structural co-factor of the tubulin molecule and render improbable the possibility that it has a regulatory role in microtubule assembly.
来自中国仓鼠卵巢（CHO）细胞的放射性标记微管蛋白可以通过与非放射性猪脑微管蛋白共聚合来分离。CHO提取物中75%的可溶性微管蛋白通过几个循环与猪蛋白共聚合，CHO或猪亚基均无优先损失。对共聚合微管进行磷酸纤维素层析后，通过SDS-聚丙烯酰胺凝胶电泳判断，CHO微管蛋白在放射化学上是均匀的。以这种方式纯化的CHO微管蛋白每摩尔蛋白质有1摩尔核苷酸以非共价键结合在不可交换或N位点。该层析表明N位点核苷酸完全是核糖鸟苷三磷酸（rGTP）。标记和追踪实验表明，在一代时间为17小时的生长细胞中，N位点GTP在细胞内交换的半衰期为33小时，而微管蛋白多肽降解的半衰期为48小时。可以检测到N位点核苷酸的γ-磷酸在细胞内的水解，但水解非常缓慢，半衰期为24小时。这些结果表明，N位点核苷酸在体内可能作为微管蛋白分子的稳定结构辅助因子发挥作用，并且其在微管组装中具有调节作用的可能性不大。
