Lobert S, Frankfurter A, Correia J J
School of Nursing, University of Mississippi Medical Center, Jackson 39216, USA.
Biochemistry. 1995 Jun 27;34(25):8050-60. doi: 10.1021/bi00025a011.
Vinblastine is an antimitotic drug that inhibits microtubule assembly and induces the self-association of tubulin into coiled spiral aggregates. Previous quantitative binding and sedimentation velocity results have been interpreted by a mechanism involving isodesmic ligand-mediated plus ligand-facilitated self-association [Na, G., & Timasheff, S. N. (1986) Biochemistry 25, 6214-6222, 6222-6228]. In this study, the vinblastine-induced self-association of porcine brain tubulin has been compared in the presence of 50 microM GDP or 50 microM GTP to investigate the role of nucleotides. Experiments at 1-4 microM tubulin in 10 mM Pipes, 1 mM MgSO4, 2 mM EGTA (pH 6.9), and varying concentrations of vinblastine (0.05-70 microM) demonstrate that GDP enhances self-association by 2-4-fold over GTP. In the presence of GDP or GTP, sedimentation velocity data can be best fit by either an indefinite ligand-mediated model or an indefinite ligand-mediated plus ligand-facilitated model. The association constant, K2, for the vinblastine-tubulin complex binding to a polymer is larger when GDP is present, while the association constant, K1, for the binding of vinblastine to tubulin heterodimers is identical in the presence of either nucleotide. The enhancement of K2 by GDP is confirmed by micropartition binding experiments with [3H]vinblastine. The fitting of sedimentation velocity and binding studies gives parameters for the interaction of vinblastine with GTP-tubulin that are identical, within error, to the previous results of Na and Timasheff. van't Hoff analysis of multiple temperature data reveals that this enhancement in the presence of GDP is due to a change in the enthalpy of self-association. Additional results suggest that the interaction of vinblastine with tubulin is identical for all beta-isotypes. Sedimentation velocity experiments in the presence of GDP or GTP show that the vinblastine-induced association of affinity-purified alpha,beta-class III tubulin is identical to that of unfractionated tubulin, although there is a difference in the abilities of unfractionated tubulin and alpha,beta III-tubulin to associate into taxol-stabilized microtubules.
长春碱是一种抗有丝分裂药物，它能抑制微管组装，并诱导微管蛋白自缔合形成盘绕的螺旋聚集体。先前的定量结合和沉降速度结果是通过一种涉及等键配体介导加配体促进自缔合的机制来解释的[Na, G., & Timasheff, S. N. (1986) Biochemistry 25, 6214 - 6222, 6222 - 6228]。在本研究中，比较了在存在50 microM GDP或50 microM GTP的情况下长春碱诱导的猪脑微管蛋白自缔合，以研究核苷酸的作用。在10 mM Pipes、1 mM MgSO4、2 mM EGTA（pH 6.9）中，对1 - 4 microM微管蛋白以及不同浓度的长春碱（0.05 - 70 microM）进行的实验表明，GDP使自缔合增强的程度比GTP高2 - 4倍。在存在GDP或GTP的情况下，沉降速度数据可以用无限配体介导模型或无限配体介导加配体促进模型进行最佳拟合。当存在GDP时，长春碱 - 微管蛋白复合物与聚合物结合的缔合常数K2更大，而在存在任何一种核苷酸的情况下，长春碱与微管蛋白异二聚体结合的缔合常数K1是相同的。用[3H]长春碱进行的微分配结合实验证实了GDP对K2的增强作用。沉降速度和结合研究的拟合给出了长春碱与GTP - 微管蛋白相互作用的参数，在误差范围内与Na和Timasheff先前的结果相同。对多个温度数据进行的范特霍夫分析表明，在存在GDP的情况下这种增强是由于自缔合焓的变化。其他结果表明，长春碱与所有β - 同型的微管蛋白的相互作用是相同的。在存在GDP或GTP的情况下进行的沉降速度实验表明，长春碱诱导的亲和纯化的α,β - III类微管蛋白的缔合与未分级微管蛋白的缔合相同，尽管未分级微管蛋白和α,β - III微管蛋白缔合形成紫杉醇稳定微管的能力存在差异。
