Hamel E, Lustbader J, Lin C M
Biochemistry. 1984 Oct 23;23(22):5314-25. doi: 10.1021/bi00317a033.
Four analogues of guanosine 5'-triphosphate (GTP) (dGTP, 3'-deoxy-GTP, arabinosyl-GTP, and 2',3'-dideoxy-GTP), which support more rapid and extensive microtubule assembly than GTP, were hydrolyzed more rapidly than GTP in reaction mixtures containing tubulin plus microtubule-associated proteins (MAPs). As with GTP, hydrolysis of the four analogues was initially closely coupled to the onset of polymerization and continued at a slower rate at the turbidity plateau. Relative to GTP, however, these analogues (and the cognate GDP analogues), particularly 3'-deoxy-GTP and 2',3'-dideoxy-GTP, bound poorly to tubulin and had a reduced ability to displace bound radiolabeled GDP under nonpolymerizing reaction conditions. Despite their reduced binding to the tubulin dimer, if polymerization occurred, all four analogues were incorporated into microtubules (as the diphosphates) in stoichiometric amounts comparable to the incorporation of GTP (in the form of GDP) with displacement of the GDP initially present in the exchangeable site. Microtubule nucleation was specifically enhanced in the presence of the analogues. With MAPs the analogues initiated microtubule assembly at temperatures 10-15 degrees C below that required by the GTP-supported reaction, and the average microtubule length was significantly reduced. In addition, MAP-independent polymerization occurred only with 2',3'-dideoxy-GTP with tubulin at 1.0 mg/mL, with the other three analogues at 2.0 mg/mL, and with GTP at 5.0 mg/mL. GTP inhibited analogue-supported polymerization at 20 degrees C with MAPs and at 37 degrees C without MAPs (tubulin, 3.5 mg/mL). Both 3'-deoxy-GTP and 2',3'-dideoxy-GTP were poor inhibitors of GTP binding and hydrolysis, but GTP potently inhibited the more vigorous hydrolysis of these analogues. We conclude that alteration of the ribose moiety reduces the affinity of a guanine nucleotide for the exchangeable site of tubulin but that a nucleotide's affinity for this site is not the major factor in its ability to support the nucleation of tubulin polymerization.
四种鸟苷 5'-三磷酸（GTP）类似物（脱氧鸟苷三磷酸、3'-脱氧-GTP、阿拉伯糖基-GTP 和 2',3'-二脱氧-GTP）比 GTP 能支持更快、更广泛的微管组装，在含有微管蛋白加微管相关蛋白（MAPs）的反应混合物中，它们比 GTP 水解得更快。与 GTP 一样，这四种类似物的水解最初与聚合反应的开始紧密相关，并在浊度平稳期以较慢的速率持续进行。然而，相对于 GTP，这些类似物（以及相应的 GDP 类似物），特别是 3'-脱氧-GTP 和 2',3'-二脱氧-GTP，与微管蛋白的结合较差，并且在非聚合反应条件下置换结合的放射性标记 GDP 的能力降低。尽管它们与微管蛋白二聚体的结合减少，但如果发生聚合反应，所有四种类似物都以化学计量的量（作为二磷酸）掺入微管中，这与 GTP（以 GDP 的形式）掺入并置换最初存在于可交换位点的 GDP 的情况相当。在类似物存在的情况下，微管成核作用特别增强。对于 MAPs，这些类似物在比 GTP 支持的反应所需温度低 10 - 15 摄氏度的温度下引发微管组装，并且平均微管长度显著缩短。此外，仅在 2',3'-二脱氧-GTP 与 1.0 mg/mL 的微管蛋白、其他三种类似物与 2.0 mg/mL 的微管蛋白以及 GTP 与 5.0 mg/mL 的微管蛋白存在时，才发生不依赖 MAPs 的聚合反应。在 20 摄氏度下，GTP 抑制 MAPs 存在时类似物支持的聚合反应，在 37 摄氏度下抑制无 MAPs 时（微管蛋白，3.5 mg/mL）的聚合反应。3'-脱氧-GTP 和 2',3'-二脱氧-GTP 都是 GTP 结合和水解的弱抑制剂，但 GTP 有力地抑制这些类似物更剧烈的水解。我们得出结论，核糖部分的改变降低了鸟嘌呤核苷酸对微管蛋白可交换位点的亲和力，但核苷酸对该位点的亲和力不是其支持微管蛋白聚合反应成核能力的主要因素。
