ROLE OF GTP HYDROLYSIS IN MICROTUBULE DYNAMICS - INFORMATION FROM A SLOWLY HYDROLYZABLE ANALOG, GMPCPP
ROLE OF GTP HYDROLYSIS IN MICROTUBULE DYNAMICS - INFORMATION FROM A SLOWLY HYDROLYZABLE ANALOG, GMPCPP
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DOI：
10.1091/mbc.3.10.1155
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发表时间：
1992-10-01
影响因子：
3.3
通讯作者：
MITCHISON, TJ
中科院分区：
文献类型：
--
作者：
HYMAN, AA;SALSER, S;MITCHISON, TJ
用GTP的类似物--鸟氨酰-(α，β)-亚甲基二磷酸盐(GMPCPP)重新研究了GTP水解物在微管动力学中的作用。该类似物结合到微管蛋白可交换核苷酸结合位点(E-Site)，亲和力比GTP低4-8倍，并促进正常微管的聚合。GMPCPP-微管蛋白的微管聚合速率与GTP-微管蛋白的聚合速率非常接近。然而，与GTP聚合的微管相比，GMPCPP-微管在等温稀释后不会迅速解聚。GMPCPP-微管的解聚速率为0.1MPCPP-微管，而GMPCPP-微管的解聚速率为500S-1。GMPCPP还完全抑制了动力不稳定性。与前人的工作相反，我们发现GMPCPP的β-伽马键进入微管晶格后，其水解率极低，其速率常数为4×10~(-7)S~(-1)。因为在聚合实验过程中GMPCPP的水解度可以忽略不计，所以可以用它来测试水解度在微管动力学中的作用。我们的结果为以下观点提供了强有力的新证据，即微管蛋白对GTP的水解不是正常聚合所必需的，而是解聚所必需的，从而是动态不稳定性的必要条件。由于GMPCPP强烈促进微管自发成核，我们认为微管蛋白对GTP的水解也具有抑制自发性微管成核的重要生物学作用。
The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta - gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 X 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.