Caplow M, Shanks J, Ruhlen R
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1994 Sep 23;269(38):23399-402.
Measurement of the affinity of microtubules for the anti-cancer drug taxol is problematic, because microtubules are not stable at the very low concentrations required to detect taxol dissociation. We have circumvented this problem by using the GTP analogue GMP-CPP (guanylyl alpha, beta-methylenediphosphonate), which renders microtubules sufficiently stable to allow binding studies with nonsaturating concentrations of taxol. AKd value equal to about 10 nM was estimated from the effect of taxol concentration on the dilution-induced disassembly rate and on the binding of [3H]taxol. With GTP-microtubules the Kd value for taxol binding by tubulin-GDP subunits in the core of the microtubule appears to be comparable with that of GMPCPP-microtubules. However, the stabilizing effect of the drug bound to tubulin subunits that arrive at ends of disassembling microtubules is attenuated by a two-step reaction sequence in which taxol dissociates (k = 30 s-1), followed by rapid (k = 1000 s-1) loss of the taxol-free tubulin subunit. This sequential reaction can be disrupted by high (micromolar) concentrations of taxol, which react rapidly with tubulin subunits at the ends of microtubules (k = 2 x 10(9) M-1 s-1). The inhibitory effect of taxol on microtubule disassembly at concentrations a thousand-fold greater than the Kd value suggests the desirability of using high taxol concentrations in chemotherapy with this compound.
测量微管与抗癌药物紫杉醇的亲和力存在问题，因为在检测紫杉醇解离所需的极低浓度下，微管不稳定。我们通过使用GTP类似物GMP-CPP（鸟苷酰α，β-亚甲基二膦酸酯）解决了这个问题，它使微管足够稳定，能够在非饱和浓度的紫杉醇下进行结合研究。根据紫杉醇浓度对稀释诱导的解聚速率和[3H]紫杉醇结合的影响，估计Kd值约为10 nM。对于GTP-微管，微管核心中微管蛋白-GDP亚基结合紫杉醇的Kd值似乎与GMPCPP-微管相当。然而，与解聚微管末端的微管蛋白亚基结合的药物的稳定作用通过两步反应序列减弱，其中紫杉醇解离（k = 30 s-1），随后是紫杉醇-free微管蛋白亚基的快速（k = 1000 s-1）损失。这种顺序反应可以被高（微摩尔）浓度的紫杉醇破坏，紫杉醇与微管末端的微管蛋白亚基快速反应（k = 2 x 10(9) M-1 s-1）。紫杉醇在比Kd值高一千倍的浓度下对微管解聚的抑制作用表明在使用该化合物进行化疗时使用高浓度紫杉醇是可取的。
