Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis.
Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis.
复制标题
在非变性电泳过程中自发形成微管蛋白二硫键交联聚集体的证据。
DOI：
10.1016/0003-9861(87)90391-2
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发表时间：
1987
影响因子：
3.9
通讯作者：
WilliamsJr,RC
中科院分区：
文献类型：
--
作者：
Correia,JJ;Welch,MK;WilliamsJr,RC
磷酸纤维素纯化的微管蛋白已显示在非变性凝胶电泳期间形成非微管聚集体的特征性“梯状”（J. J. Correia和R. J. C.小威廉姆斯（1985）Arch.Biochem.Biopolymer.239，120-129）。在本文中，我们描述的证据表明，负责形成这些低聚物颗粒的亚基间键是二硫化物。双向非变性-变性凝胶电泳结果表明，每个聚集区由微管蛋白的α-和β-亚基组成。在十二烷基硫酸钠（SDS）-电泳步骤中省略β-巯基乙醇导致出现聚集体模式，并暗示二硫键在其稳定性中。从二维凝胶的第二（SDS）维中的迁移率估计的分子量表明，聚集体以单体而不是异二聚体的单位交联。与该结论一致，单独的α-或β-亚基（通过等电聚焦分离）将形成相同的聚集体梯。微管蛋白的二硫化物交联也可在溶液中实现。高浓度的酒精、氧化剂的存在、高pH值和高温、使微管蛋白变性并引起快速非共价聚集或沉淀的条件有利于它。当在不存在β-巯基乙醇的情况下通过SDS-凝胶电泳以及通过定量巯基和二硫化物滴定监测聚集体形成作为时间的函数时，交联反应的最有效条件包括>75%的乙醇、过量的H2 O2或过量的碘。这些结果表明，在非变性凝胶电泳过程中，疏水凝胶基质的接近、高pH值、氧化剂的存在、高蛋白浓度、微管蛋白非特异性聚集的倾向以及αβ-微管蛋白中多达20个巯基的可用性有助于二硫键交联微管蛋白聚集体的自发形成。
Phosphocellulose-purified tubulin has been shown to form a characteristic “ladder” of nonmicrotubular aggregates during nondenaturing gel electrophoresis (J. J. Correia and R. C. Williams, Jr. (1985)Arch. Biochem. Biophys.239, 120–129). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of α- and β-subunits of tubulin. Omission of β-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, α- or β-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of β-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included >75% alcohol, excess H2O2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in αβ-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide-crosslinked tubulin aggregates.