KINETICS AND MECHANISM OF COLCHICINE BINDING TO TUBULIN - EVIDENCE FOR LIGAND-INDUCED CONFORMATIONAL CHANGE
KINETICS AND MECHANISM OF COLCHICINE BINDING TO TUBULIN - EVIDENCE FOR LIGAND-INDUCED CONFORMATIONAL CHANGE
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DOI：
10.1021/bi00613a024
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
GARLAND, DL
中科院分区：
文献类型：
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作者：
GARLAND, DL
[猪脑]微管蛋白-秋水仙碱复合物形成的动力学进行了重新研究，使用同位素标记和荧光技术。结合的时间过程由一个相对较快的步骤组成，该步骤负责大部分反应，随后是一个较慢的步骤，该步骤占结合秋水仙碱的5-10%。慢步的起源尚不清楚。快速步骤的分析表明，在伪一阶条件下，速率常数协会，kobsd，增加秋水仙素和微管蛋白浓度线性增加，然后偏离线性在高浓度。（kobsd-k-2）-1 vs.浓度-1曲线呈线性。可以解释数据的最简单机制是：**图形 **。动力学常数由计算机曲线拟合得到。由此得到快速平衡步骤的平衡常数K1 = 6 × 10 - 4。103 M-1;k2 =（2-3）×103 s-1和k-2 =（5-9）×10-6 s-1条件下通过同位素标记法测定的微管蛋白-秋水仙碱复合物解离的速率常数为5.3 ×10-6 s-1条件下因此，k-2是秋水仙素从微管蛋白-秋水仙素复合物中解离的限速步骤。使用蔗糖改变粘度来确定kobsd对溶剂粘度的依赖性。在低浓度的秋水仙素的表观二阶速率常数随粘度的增加而降低，而在高浓度的秋水仙素的粘度的速率依赖性显着降低。当秋水仙素结合微管蛋白时观察到的荧光增强是秋水仙素诱导的构象变化步骤的结果。通过聚合-解聚循环制备的微管蛋白被分离成2 MW物质，峰1包含高MW物质，峰2仅由微管蛋白二聚体组成。在两个峰中秋水仙碱与微管蛋白结合的表观二级速率常数基本相同，分别为1.7和1.4 × 10 - 6。102 M-1 s-1。当微管蛋白与秋水仙素预孵育后进行色谱分析时，结合的秋水仙素仅在峰2中作为微管蛋白二聚体发现。
The kinetics of [porcine brain] tubulin-colchicine complex formation were reinvestigated using both isotopic labeling and fluorescence techniques. The time course for association is composed of a relatively fast step, which is responsible for most of the reaction, followed by a slower step, which accounted for 5-10% of the bound colchicine. The origin of the slow step is not known. Analysis of the fast step showed that under pseudo-1st-order conditions, rate constants for association, kobsd, increased linearly with both increasing colchicine and tubulin concentration and then deviated from linearity at high concentrations. Plots of (kobsd-k-2)-1 vs. concentration-1 were linear. The simplest mechanism that can explain the data is: .**GRAPHIC**. Kinetic constants were obtained by computer curve fitting of the data. These yield an equilibrium constant for the rapid equilibrium step K1 = 6 .times. 103 M-1;k2 = (2-3) .times. 103 s-1 and k-2 = (5-9) .times. 10-6 s-1. The rate constant for the dissociation of the tubulin-colchicine complex determined by the isotopic labeling method was 5.3 .times. 10-6 s-1. Therefore, k-2 is the rate-limiting step for colchicine dissociation from the tubulin-colchicine complex. The dependency of kobsd on solvent viscosity was determined using sucrose to vary the viscosity. At low colchicine concentrations the apparent 2nd-order rate constant is decreased with increasing viscosity, whereas at high colchicine concentrations the rate dependency on viscosity is significantly reduced. The fluorescence enhancement observed when colchicine binds to tubulin is a consequence of the colchicine-induced conformational change step. Tubulin prepared by cycles of polymerization-depolymerization was separated into 2 MW species, peak 1 containing the high-MW species and peak 2 consisting only of tubulin dimers. The apparent 2nd-order rate constants for association of colchicine to tubulin in both peaks were essentially the same, 1.7 and 1.4 .times. 102 M-1 s-1, respectively. When tubulin was preincubated with colchicine prior to chromatography, bound colchicine was only found in peak 2 as the tubulin dimer.