ASSEMBLY OF PURIFIED GDP TUBULIN INTO MICROTUBULES INDUCED BY TAXOL AND TAXOTERE - REVERSIBILITY, LIGAND STOICHIOMETRY, AND COMPETITION
ASSEMBLY OF PURIFIED GDP TUBULIN INTO MICROTUBULES INDUCED BY TAXOL AND TAXOTERE - REVERSIBILITY, LIGAND STOICHIOMETRY, AND COMPETITION
复制标题
DOI：
10.1021/bi00062a003
复制
发表时间：
1993-03-23
期刊：
影响因子：
2.9
通讯作者：
ANDREU, JM
中科院分区：
文献类型：
--
作者：
DIAZ, JF;ANDREU, JM
通过一种新的直接核苷酸交换方法制备了在可交换核苷酸结合位点与GDP完全配体的纯化微管蛋白。这种通常无活性的GDP-微管蛋白通过抗肿瘤药物紫杉醇或其更可溶的侧链类似物泰索帝在含Mg 2+的缓冲液中的结合而被驱动组装成微管，并且通过冷却溶液而分解。因此，这种配体诱导的平衡微管组装系统免除了在微管蛋白的核苷酸位点处结合的γ-磷酸盐-金属阳离子配体的需要。GDP-微管蛋白也可以形成双环的特征性伪有序聚集体。这些聚集体在加热或通过添加GTP而解离。核苷酸γ-磷酸的回代允许甘油诱导的组装而不需要紫杉醇，并将药物诱导的微管组装所需的临界蛋白质浓度降低2.6 +/-0。1.配体诱导的组装在与微管蛋白等摩尔的紫杉醇或泰索帝浓度下最大，并且两种药物以0.99 +/-0.04配体/α二聚体的化学计量结合组装的微管蛋白。泰索帝显然与紫杉醇竞争相同的结合位点，有效亲和力大1.9 +/-0.1倍。类似地，紫杉醇诱导的GDP-微管蛋白或GTP-微管蛋白的组装以比紫杉醇小2.1 +/-0.1倍的临界蛋白浓度进行。
Purified tubulin fully liganded to GDP at the exchangeable nucleotide binding site has been prepared by a new direct nucleotide exchange procedure. This normally inactive GDP-tubulin is driven to assemble into microtubules by the binding of the antitumor drug taxol or its more soluble side-chain analogue Taxotere in Mg2+-containing buffer, and it disassembles by cooling the solution. Therefore this ligand-induced equilibrium microtubule assembly system dispenses with the requirement of a gamma-phosphate-metal cation ligand bound at the nucleotide site for tubulin to be active. GDP-tubulin can also form characteristic pseudoordered aggregates of double rings. These aggregates dissociate upon warming or by addition of GTP. Back-substitution of the nucleotide gamma-phosphate permits glycerol-induced assembly without taxol and reduces the critical protein concentration required for drug-induced microtubule assembly by a factor of 2.6 +/- 0. 1. The ligand-induced assembly is maximal at taxol or Taxotere concentrations equimolar with tubulin, and both drugs bind to assembled tubulin with a stoichiometry of 0.99 +/- 0.04 ligand per alphabeta dimer. Taxotere apparently competes with taxol for the same binding site, with 1.9 +/- 0.1 times larger effective affinity. Similarly, the Taxotere-induced assembly of GDP-tubulin or GTP-tubulin proceeds with a critical protein concentration 2.1 +/- 0.1 times smaller than with taxol.