Grover S, Hamel E
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Eur J Biochem. 1994 May 15;222(1):163-72. doi: 10.1111/j.1432-1033.1994.tb18854.x.
Microtubule-associated-protein-dependent assembly of tubulin with GDP in the exchangeable site (tubulin-GDP) can occur with minimal free Mg2+ (< 3 microM). This reaction is totally inhibited by EDTA and by GTP concentrations over 2 mM and stimulated by MgCl2. Quantitative aspects of this stimulation are affected by both the Mg2+ and GTP concentrations but no relationship exists between reaction rates and relative amounts of different magnesium and GTP species. GTP binding to tubulin-GDP, while maximally stimulated 2-3-fold by exogenous MgCl2, was inhibited less than 50% by EDTA, and the amount of GTP bound increased as its concentration rose to levels that inhibited polymerization. Studies on the binding of Mg2+ to tubulin-GDP in the presence and absence of GTP showed that the increase in the amount of tubulin-associated Mg2+ was substoichiometric to the amount of GTP bound (maximum stoichiometry of additional Mg2+ to GTP bound, 0.7). Upon polymerization the increased Mg2+ content of tubulin was reduced, indicating its loss during GTP hydrolysis. Mg2+ thus plays a critical role in assembly distinct from its enhancement of GTP binding to the exchangeable site. If magnesium is present in trace amounts, this role must either be catalytic during polymerization or limited to nucleation.
微管蛋白与可交换位点的GDP（微管蛋白 - GDP）在微管相关蛋白依赖下的组装，在游离Mg2+极少（<3 microM）的情况下即可发生。该反应完全被EDTA和超过2 mM的GTP浓度抑制，并被MgCl2刺激。这种刺激的定量方面受Mg2+和GTP浓度两者影响，但反应速率与不同镁和GTP种类的相对量之间不存在关联。GTP与微管蛋白 - GDP的结合，虽然在外源MgCl2作用下最大可被刺激2 - 3倍，但被EDTA抑制不到50%，并且随着GTP浓度升至抑制聚合的水平，结合的GTP量增加。在有和没有GTP的情况下对Mg2+与微管蛋白 - GDP结合的研究表明，与微管蛋白结合的Mg2+量的增加与结合的GTP量呈亚化学计量关系（额外Mg2+与结合的GTP的最大化学计量比为0.7）。聚合时，微管蛋白增加的Mg2+含量减少，表明其在GTP水解过程中损失。因此，Mg2+在组装中起着关键作用，这与其增强GTP与可交换位点的结合不同。如果镁以痕量存在，该作用要么在聚合过程中起催化作用，要么仅限于成核作用。
