Ross Jennifer L, Fygenson D Kuchnir
Physics Department, University of California, Santa Barbara, California 93106-9530, USA.
Biophys J. 2003 Jun;84(6):3959-67. doi: 10.1016/S0006-3495(03)75123-6.
Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With >63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k(off). It was found that 10k(off)(GMPCPP) approximately equal k(off)(GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With <63% sites occupied, recovery times decreased as approximately Tax for both types of microtubules. We conclude that the diffusion of taxol inside the microtubule bundle is hindered by rebinding events when open sites are within approximately 7 nm of each other.
利用光漂白后荧光恢复技术，在流动排列的微管束上研究了紫杉醇在微管内的移动性。微管束由微管组成，微管蛋白二聚体上可交换位点处分别含有鸟苷-5'-三磷酸-3'-O-(2'-甲基)亚磷酸酯（GMPCPP）或鸟苷三磷酸（GTP）。恢复时间对微管束的厚度和堆积情况敏感，这表明紫杉醇分子能够在微管束中横向移动。对于含有GMPCPP的样品，通过控制溶液中紫杉醇的浓度来改变沿微管开放结合位点的密度。当占据的位点>63%时，恢复时间与紫杉醇浓度无关，因此与微观解离速率k(off)成反比。研究发现，10k(off)(GMPCPP)约等于k(off)(GTP)，这与紫杉醇在GMPCPP和GTP微管上平衡常数的差异一致，但并不完全能解释该差异。当占据的位点<63%时，两种类型微管的恢复时间均随Tax下降。我们得出结论，当开放位点彼此距离约7 nm以内时，微管内紫杉醇的扩散会受到再结合事件的阻碍。
