Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source
Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source
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DOI：
10.1091/mbc.e01-10-0089
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发表时间：
2002-06-01
影响因子：
3.3
通讯作者：
Fee, L
中科院分区：
文献类型：
--
作者：
Caplow, M;Fee, L
研究发现，在没有蛋白质辅助因子的情况下，微管蛋白二聚体（α - β + α ' α ' α ' α ' β + α ' α）之间的微管蛋白亚基交换不会发生，GTP水解与纯微管蛋白二聚体和单体处于快速平衡的假设相冲突。这个假设是二聚体解离的K-d的许多物理化学测量的基础。为了解决这种差异，我们使用表面等离子体共振来确定二聚体解离的速率常数。微管蛋白- gtp的解离半衰期为9.6 h，微管蛋白- gdp的解离半衰期为2.4 h，无核苷酸的解离半衰期为1.3 h。K-d等于10(-11)M，由测量的解离速率和估计的缔合速率计算得到。二聚体解离是可逆的，二聚体的形成不需要GTP水解或蛋白质辅助因子的折叠信息，因为0.2 muM微管蛋白- gdp在培养20小时后被洗脱为二聚体。因为20小时对应于解离的8个半倍，如果解离是不可逆反应，并且二聚体的形成需要GTP或蛋白质辅助因子，则只存在单体。从0.02-2 nM微管蛋白- gdp的凝胶排斥色谱研究中获得了10(-11)M K-d的额外证据。微管蛋白二聚体的缓慢解离表明，蛋白微管蛋白辅助因子的作用是催化二聚体解离，而不是二聚体的组装。假设N-site-GTP解离来自单体，我们的结果与体外N-site-GTP的半衰期为16小时和微管蛋白N-site-GTP在CHO细胞中的半衰期为33小时一致。
The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K-d for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was similar to9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A K-d equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 muM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K-d was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.