COLD DEPOLYMERIZATION OF MICROTUBULES TO DOUBLE RINGS - GEOMETRIC STABILIZATION OF ASSEMBLIES
COLD DEPOLYMERIZATION OF MICROTUBULES TO DOUBLE RINGS - GEOMETRIC STABILIZATION OF ASSEMBLIES
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DOI：
10.1021/bi00449a028
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发表时间：
1989-11-14
期刊：
影响因子：
2.9
通讯作者：
TIMASHEFF, SN
中科院分区：
文献类型：
--
作者：
MELKI, R;CARLIER, MF;TIMASHEFF, SN
已经检测了0 ℃下微管解聚的动力学途径。在[3 H]GDP或[3 H]GTP或痕量125 I二聚微管蛋白存在下，在0 ℃下解聚由含MAP和不含MAP的微管蛋白制成的微管。解聚的产物在柱上分离，它们的结构通过电子显微镜进行鉴定，并确定在系统的不同组分中掺入3 H或125 I标记的时间过程。在非微管状态中发现的微管蛋白的两种主要组装状态是α-。β的二聚体和双环。动力学数据表明，从拆卸微管环的形成不会发生直接卷曲的原丝，如以前认为的，但拆卸GDP亚基是在非常快速的平衡与弯曲的低聚物，动力学中间体的GDP-微管蛋白的等键组装。在低至10 μ M的浓度下，由二聚体形成寡聚体和环比α-聚体上的核苷酸交换快得多。贝塔微管蛋白相反，双环的交换比α-环上的核苷酸交换慢。贝塔微管蛋白，在不存在MAP的情况下增加1个数量级，在存在MAP的情况下增加2个数量级。这些结果支持先前提出的模型来解释自发振荡的微管组装。它们与微管蛋白的两种构象之间的平衡的存在是一致的，“直的”，即，微管形成和“弯曲”，即，在结合核苷酸的变构控制下形成环。直链构象需要在γ-羟基上存在两个可电离的羟基。GTP或GDP-Pi中的磷酸盐。
The kinetic pathway of microtubule depolymerization at 0.degree.C has been examined. Microtubules made of MAP-containing and MAP-free tubulins were depolymerized at 0.degree.C in the presence of [3H]GDP or [3H]GTP or of trace amounts of 125I dimeric tubulin. The products of depolymerization were separated on a column, their structures were identified by electron microscopy, and the time course of incorporation of 3H or 125I labels in the different components of the system was determined. Two predominant assembly states of tubulin found in the nonmicrotubule state were .alpha.-.beta. dimers and double rings. Kinetic data indicate that ring formation from disassembling microtubules does not occur by direct coiling of protofilaments as previously thought, but disassembling GDP subunits are in very rapid equilibrium with curved oligomers that are kinetic intermediates in the isodesmic assembly of GDP-tubulin. The formation of oligomers and rings from dimers, at concentrations as low as 10 .mu.M, is much faster than nucleotide exchange on .alpha.-.beta.-tubulin. Disassembly of double rings, in contrast, is slower than nucleotide exchange on .alpha.-.beta.-tubulin, by 1 order of magnitude in the absence of MAPs and 2 orders of magnitude in the presence of MAPs. These results support the model proposed previously to explain spontaneous oscillations in microtubule assembly. They are consistent with the existence of an equilibrium between two conformations of tubulin, "straight", i.e., microtubule forming, and "curved", i.e., ring forming, under the allosteric control of bound nucleotide. The straight conformation requires the presence of two ionizable hydroxyls on the .gamma.-phosphate in GTP or GDP-Pi.