Luyckx M, Brunet C, Cazin M
Methods Find Exp Clin Pharmacol. 1984 Nov;6(11):679-84.
We have evaluated the effects that methodology (incubation time, concentration of beef brain tubulin) have on the measurement of the interaction of tubulin with colchicine. The results show that, with DEAE filter paper assay, binding is maximal for 3 hr of incubation at 37 degrees C, and for low concentrations of tubulin (0.05 mg/ml). In these conditions binding constants determined with SCATCHARD plot are: constant of dissociation (KD) of complex tubulin-colchicine equal to 9.8 X 10(-7) M and stechiometry equal to 0.57 mole of colchicine bound for 1 mole of tubulin dimer. Competition of drugs with colchicine binding site may be evaluated with this methodology. IC50 and affinity constants (Ki) of podophyllotoxin, a competitive inhibitor of colchicine-binding determined with these experimental conditions were 1.3 X 10(-6) M and 1.1 X 10(-6) M, respectively.
我们评估了方法学因素（孵育时间、牛脑微管蛋白浓度）对微管蛋白与秋水仙碱相互作用测量的影响。结果表明，采用DEAE滤纸分析法时，在37℃孵育3小时以及微管蛋白浓度较低（0.05mg/ml）的情况下结合作用最强。在这些条件下，用斯卡查德图测定的结合常数为：微管蛋白 - 秋水仙碱复合物的解离常数（KD）等于9.8×10⁻⁷M，化学计量比为每1摩尔微管蛋白二聚体结合0.57摩尔秋水仙碱。用该方法可评估药物与秋水仙碱结合位点的竞争情况。在这些实验条件下测定的秋水仙碱结合竞争性抑制剂鬼臼毒素的IC50和亲和常数（Ki）分别为1.3×10⁻⁶M和1.1×10⁻⁶M。
