Duanmu C, Lin C M, Hamel E
Biochim Biophys Acta. 1986 Mar 19;881(1):113-23. doi: 10.1016/0304-4165(86)90104-2.
Glycerol-induced tubulin polymerization supported by non-guanine nucleotides was examined. The electrophoretically homogeneous tubulin was devoid of nucleoside diphosphate kinase activity and 95% saturated with exchangeable GDP and nonexchangeable GTP. All purine ribonucleoside 5'-triphosphates were active but no polymerization occurred with CTP or UTP. All polymerization reactions, as a function of nucleotide concentration, were similar: above a minimum (threshold) concentration, as the amount of nucleotide increased the reaction became progressively more rapid and extensive with a progressively shorter nucleation period. Threshold concentrations of ATP, XTP, ITP and GTP were 0.6 mM, 0.3 mM, 30 microM and 7 microM, respectively. Most ribose- and polyphosphate-modified ATP analogs also supported polymerization at high concentrations, but the activity of these analogs relative to ATP was very similar to the activity of cognate GTP analogs relative to GTP. Polymerization with ATP was associated with an ATPase reaction. ATP hydrolysis was potently inhibited by GDP and GTP and altered by antimitotic drugs in parallel with the effects of these agents on GTP hydrolysis. Substantial amounts of [8-14C]GDP bound in the exchangeable site of tubulin were displaced during polymerization with GTP or ATP, but much higher concentrations of ATP were required for equivalent displacement of the tubulin-bound GDP. Polymerization with GTP or ATP was inhibited in a qualitatively similar manner by GDP, with increasing concentrations of GDP causing a progressive prolongation of the nucleation period and reduction in reaction rate and extent. However, complete inhibition of polymerization required that GDP:GTP much greater than 1, but that GDP:ATP much less than 1. Inhibition appeared to be primarily competitive, since with higher triphosphate concentrations higher GDP concentrations were required for comparable inhibition. We conclude that ATP effects on tubulin polymerization are mediated through a feeble interaction at the exchangeable GTP site.
研究了由非鸟嘌呤核苷酸支持的甘油诱导的微管蛋白聚合反应。经电泳均一的微管蛋白缺乏核苷二磷酸激酶活性，且95%被可交换的GDP和不可交换的GTP饱和。所有嘌呤核糖核苷5'-三磷酸均有活性，但CTP或UTP不能引发聚合反应。所有聚合反应，作为核苷酸浓度的函数，表现相似：在最低（阈值）浓度以上，随着核苷酸量的增加，反应逐渐变得更快、更广泛，成核期逐渐缩短。ATP、XTP、ITP和GTP的阈值浓度分别为0.6 mM、0.3 mM、30 μM和7 μM。大多数核糖和多磷酸盐修饰的ATP类似物在高浓度时也支持聚合反应，但这些类似物相对于ATP的活性与同源GTP类似物相对于GTP的活性非常相似。ATP引发的聚合反应与ATP酶反应相关。GDP和GTP能有效抑制ATP水解，抗有丝分裂药物可改变ATP水解，且这些药物对ATP水解的影响与对GTP水解的影响平行。在与GTP或ATP聚合反应过程中，大量结合在微管蛋白可交换位点的[8-14C]GDP被置换，但等量置换微管蛋白结合的GDP需要更高浓度的ATP。GDP以定性相似的方式抑制GTP或ATP引发的聚合反应，随着GDP浓度增加，成核期逐渐延长，反应速率和程度降低。然而，完全抑制聚合反应要求GDP:GTP远大于1，但GDP:ATP远小于1。抑制作用似乎主要是竞争性的，因为在更高的三磷酸浓度下，需要更高的GDP浓度才能产生可比的抑制作用。我们得出结论，ATP对微管蛋白聚合的影响是通过在可交换GTP位点的微弱相互作用介导的。
