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首页> 外文OA文献 >Mechanism of tubulin-colchicine recognition: a kinetic study of the binding of the colchicine analogues colchicide and isocolchicine.
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Mechanism of tubulin-colchicine recognition: a kinetic study of the binding of the colchicine analogues colchicide and isocolchicine.

机译：微管蛋白-秋水仙碱识别的机制：秋水仙碱类似物杀虫剂和异秋水仙碱结合的动力学研究。

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摘要

Colchicide (IDE) is a colchicine (COL) analogue in which the C-10 methoxy group is replaced by a hydrogen atom. Its binding to tubulin is accompanied by a quenching of the protein fluorescence. The fluorescence decrease shows a monoexponential time dependence. The observed rate constant increases in a non-linear way with the total concentration of IDE, allowing the determination of a binding constant for an initial binding site (K1=5300+/-300 M-1) and the rate constant for the subsequent isomerization (k2=0.071+/-0.002 s-1) at 25 degrees C. The rate constant, k-2, for the reversed isomerization can be determined by displacement experiments. Despite the minor alteration of the C-ring substituent, the kinetic and thermodynamic parameters of binding are substantially different from those of COL itself, for both steps. In isocolchicine (ISO) the carbonyl oxygen atom and the methoxy groups of the C-ring have been interchanged. Its binding to tubulin only results in small fluorescence and absorbance changes. Therefore competition experiments with MTC [2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4, 6-cycloheptatrien-1-one] were performed. ISO competes rapidly and with low affinity with MTC. Fluorimetric titrations of tubulin with MDL (MDL 27048 or trans-1-(2,5 dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-propen-1 -one) in the presence and absence of ISO give evidence for the existence of a second, slow-reacting low-affinity site for ISO that is not accessible to MTC or MDL. The relevance of these results for the recognition of COL is analysed.
机译：杀菌剂（IDE）是一种秋水仙碱（COL）类似物，其中C-10甲氧基被氢原子取代。它与微管蛋白的结合伴随着蛋白质荧光的猝灭。荧光下降显示出单指数时间依赖性。观察到的速率常数随IDE的总浓度呈非线性增加，从而可以确定初始结合位点（K1 = 5300 +/- 300 M-1）的结合常数和随后异构化的速率常数（k2 = 0.071 +/- 0.002 s-1）在25摄氏度时。反向异构化的速率常数k-2可以通过置换实验确定。尽管C环取代基的变化很小，但在两个步骤中，结合的动力学和热力学参数都与COL自身的参数基本不同。在异秋水仙碱（ISO）中，羰基氧原子和C环的甲氧基已互换。它与微管蛋白的结合只会导致较小的荧光和吸光度变化。因此，进行了MTC [2-甲氧基-5-（2'，3'，4'-三甲氧基苯基）-2,4,6-环庚三烯-1-酮]的竞争实验。 ISO竞争迅速，与MTC的亲和力很低。在存在和不存在以下条件下，用MDL（MDL 27048或反式1-（2,5二甲氧基苯基）-3- [4-（二甲基氨基）苯基] -2-甲基-2-丙烯-1-酮）进行微管蛋白的荧光滴定。 ISO提供了证据，表明存在另一个MTC或MDL无法访问的ISO慢反应低亲和力位点。分析了这些结果与识别COL的相关性。

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