Taxol binds to polymerized tubulin in vitro.
Taxol binds to polymerized tubulin in vitro.
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DOI：
10.1083/jcb.91.2.479
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发表时间：
1981-11
期刊：
影响因子：
--
通讯作者：
Horwitz SB
中科院分区：
文献类型：
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作者：
Parness J;Horwitz SB
紫杉醇是一种天然植物产物，能提高体外微管组装的速率和程度，并能稳定体外和细胞内的微管。用沉降法研究了[3 H]紫杉醇与微管蛋白的结合。在[（3）H]紫杉醇存在下组装的微管与药物特异性结合，表观结合常数K（app）为8.7 × 19（-7）M，结合饱和，计算的最大结合比B（max）为0.6 mol紫杉醇结合/mol微管蛋白二聚体。[（3）H]紫杉醇也结合和组装磷酸纤维素纯化的微管蛋白，我们认为紫杉醇稳定导致微管聚合物形成的二聚体之间的相互作用。对于微管蛋白和磷酸纤维素纯化的微管蛋白，结合饱和发生在与微管蛋白二聚体浓度的近似化学计量。在组装条件下，鬼臼毒素和长春碱以一种复杂的方式抑制[（3）H]紫杉醇与微管蛋白的结合，我们认为这反映了这些药物之间的竞争，不是针对单个结合位点，而是针对微管蛋白的不同形式（二聚体和多聚体）。用GTP或5 '-鸟苷酰-α，β-亚甲基二膦酸酯（GPCPP）组装的稳态微管，GPCPP是一种GTP类似物，据报道可抑制微管的螺旋研磨（I. V.桑多瓦尔和K.韦伯1980. J.Biol.Chem.255：6966-6974），以与在[（3）H]紫杉醇存在下组装的微管大致相同的化学计量结合[（3）H]紫杉醇。这些数据表明，紫杉醇结合位点存在于完整的微管。未标记的紫杉醇竞争性地从微管中置换[（3）H]紫杉醇，而鬼臼毒素、长春碱和CaCl（2）则不能。然而，鬼臼毒素和长春碱减少了沉淀的紫杉醇稳定的微管的质量，但是颗粒中结合的[（3）H]紫杉醇的比活性保持恒定。我们的结论是，紫杉醇结合特异性和可逆的微管蛋白的化学计量接近统一的聚合形式。
Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.