Department of Developmental and Cell Biology, University of California, 92717, Irvine, CA, USA.
Planta. 1987 Feb;170(2):230-41. doi: 10.1007/BF00397893.
The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K i) of 1.4·10(-4) M for rose tubulin and an apparent K i=8.8·10(-7) M for brain tubulin. The binding of [(3)H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2-3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [(3)H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10(2) M(-1) and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10(6) M(-1) and r=0.45 at 37°C. The binding of [(3)H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.
从玫瑰（Rosa. sp. cv. Paul's scarlet）培养细胞中抑制微管蛋白聚合的秋水仙碱和秋水仙碱与微管蛋白的结合在体外进行了检查，并与牛脑微管蛋白平行实验获得的数据进行了比较。微管蛋白聚合的浊度测量在低微管蛋白浓度下是敏感和半定量的，并且符合丝状螺旋聚合物组装的成核和缩合聚合机制的一些特征。秋水仙碱在 24°C 时抑制聚合的快速相，对玫瑰微管蛋白的表观抑制常数（K i）为 1.4·10（-4）M，对脑微管蛋白的表观 K i=8.8·10（-7）M。[(3)H]秋水仙碱与玫瑰微管蛋白结合形成微管蛋白-秋水仙碱复合物的结合对温度有轻微依赖性且缓慢，在 24°C 下达到平衡需要 2-3 小时，并且不受硫酸长春碱影响。[(3)H]秋水仙碱与玫瑰微管蛋白的结合是饱和的，Scatchard 分析表明存在一个低亲和力结合位点，其表观亲和力常数（K）为 9.7·10（2）M（-1），在 24°C 时估计摩尔结合比（r）为 0.47。脑微管蛋白的 K 值为 2.46·10（6）M（-1），r 值为 0.45，在 37°C 时。[(3)H]秋水仙碱与玫瑰微管蛋白的结合受过量未标记秋水仙碱的抑制，但不受鬼臼毒素或三羟甲基噁唑烷酮的抑制。数据表明植物和动物微管蛋白上的秋水仙碱结合位点存在差异，并表明植物微管蛋白聚合对秋水仙碱的相对抗性是由于秋水仙碱与微管蛋白的低亲和力相互作用所致。
