Determination of the size and chemical nature of the stabilizing "cap" at microtubule ends using modulators of polymerization dynamics
Determination of the size and chemical nature of the stabilizing "cap" at microtubule ends using modulators of polymerization dynamics
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DOI：
10.1021/bi011767m
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发表时间：
2002-02-05
期刊：
影响因子：
2.9
通讯作者：
Wilson, L
中科院分区：
文献类型：
--
作者：
Panda, D;Miller, HP;Wilson, L
微管（MT）末端稳定帽的大小和化学性质仍然是个谜，很大程度上是因为很难直接检测和测量它。通过用微量的[γ（32）P]GTP脉冲牛脑微管（MT）的稳态悬浮液，并通过50%蔗糖垫沉淀MT以将背景污染P-32降低到可忽略的水平，我们能够检测到少量的P-32分子，其保持稳定地结合到MT（平均25.5个P-32分子/MT）。通过薄层色谱法分析稳定结合的P-32的化学形式表明，它都是P-32-正磷酸盐（P-32（i））。32 P（i）被确定位于MT末端，因为秋水仙碱和长春碱（通过特异性结合MT末端抑制微管蛋白掺入NIT的药物）减少了稳定结合的P-32（i）的量。紫杉醇是一种通过沿着MT表面而不是末端结合来稳定MT动力学的药物，它不影响结合的P-32（i）的化学计量。如果结合的P-32平均分布在两端，则每端将含有大约12 -13个P-32（i）分子。氟化铍（BeF 3-）和氟化铝（AlF 4-），无机磷酸盐类似物，抑制了单个MT的动态不稳定行为，从而使其稳定。例如，BeF 3-（70 μ M）使MT缩短速率降低2.5倍，并使从生长或衰减状态到快速缩短的转变频率降低2倍。这些数据支持MT末端的稳定帽由单层微管蛋白GDP-P-i亚基组成的假设。该数据还支持以下假设：产生不稳定的GDP-微管蛋白核心的机制涉及β 1的释放而不是GTP的水解。
The size and chemical nature of the stabilizing cap at microtubule (MT) ends has remained enigmatic, in large part because it has been difficult to detect and measure it directly. By pulsing steady-state suspensions of bovine brain microtubules (MTs) with trace quantities of [gamma(32)P]GTP and sedimenting the MTs through 50% sucrose cushions to reduce background contaminating P-32 to negligible levels, we were able to detect a small number of P-32 Molecules that remain stably bound to the MTs (a mean of 25.5 molecules of P-32 per MT). Analysis of the chemical form of the stably bound P-32 by thin-layer chromatography revealed that it was all P-32-orthophosphate (P-32(i)). The 32P(i) was determined to be located at the MT ends because colchicine and vinblastine, drugs that suppress tubulin incorporation into the NIT by binding specifically at MT ends, reduced the quantity of the stably bound P-32(i). Taxol, a drug that stabilizes MT dynamics by binding along the MT surface rather than at the ends, did not affect the stoichiometry of the bound P-32(i). If the bound P-32 is equally distributed between the two ends, each end would contain similar to12-13 molecules of P-32(i). Beryllium fluoride (BeF3-) and aluminum fluoride AlF4-), inorganic phosphate analogues, suppressed the dynamic instability behavior of individual MTs and, thus, stabilized them. For example, BeF3- (70 muM) reduced the MT shortening rate by 2.5-fold and decreased the transition frequency from the growing or the attenuated state to rapid shortening by 2-fold. The data support the hypothesis that the stabilizing cap at MT ends consists of a single layer of tubulin GDP-P-i subunits. The data also support the hypothesis that the mechanism giving rise to the destabilized GDP-tubulin core involves release of P-i rather than hydrolysis of the GTP.