Traub P, Kühn S, Grüb S
Max-Planck-Institut für Zellbiologie, Ladenburg bei Heidelberg, Germany.
J Mol Biol. 1993 Apr 5;230(3):837-56. doi: 10.1006/jmbi.1993.1205.
Affinity chromatography on single-stranded (ss)DNA-cellulose in conjunction with gel permeation chromatography in the presence of urea was employed to separate the intermediate filament (IF) protein complement of catalytically oxidized BHK-21 cell Triton cytoskeletons into disulfide-cross-linked homo- and heterodimers of desmin and vimentin and uncross-linked homodimers. The same separation was performed on a desmin-vimentin mixture under autoxidizing conditions in 6 M-urea to obtain the respective cross-linked collision complexes of both proteins. In 5 M-urea, the oxidation products were identified as dimers that were physically indistinguishable from uncross-linked homodimers, suggesting that they were in the form of partially denatured face-to face pairs. Heterodimers derived from intact IFs were identical to those derived from collision complexes. In the presence of 2-mercaptoethanol, heterodimers were unstable and transformed spontaneously into homodimers. After removal of urea, all cross-linked dimers were totally unable to polymerize into filaments; however, in the presence of 2-mercaptoethanol they showed a normal assembly competence. This inability of oxidized homo- and heterodimers to polymerize, together with the relatively low yield of cross-linked dimers obtained from cytoskeletons, is probably due to the introduction of steric strain into the dimers by disulfide bond formation. Substantial amounts of cross-linked heterodimers could also be isolated from IFs reconstituted from mixtures of desmin and vimentin in their homodimeric or tetrameric forms. Taken together, these results suggest that the cross-linked dimers isolated from cytoskeletons arise from a reaction between subfilament strands of IFs rather than from disulfide bond formation within pre-existing dimers and that the heterotypic IFs of BHK-21 cells are largely formed from homodimers and tetramers, respectively, rather than from heterodimers. The differential capacity of desmin and vimentin to interact with ssDNA has also been exploited to distinguish between homotypic and heterotypic protofilaments, the latter consisting of one homodimer of each protein species. This distinction could be made on the basis of characteristic differences in the sedimentation behavior of the respective protein-DNA complexes.
在尿素存在的情况下，结合凝胶渗透色谱法，利用单链（ss）DNA - 纤维素亲和色谱法，将催化氧化的BHK - 21细胞Triton细胞骨架中的中间丝（IF）蛋白组分分离为结蛋白和波形蛋白的二硫键交联同二聚体和异二聚体以及未交联的同二聚体。在6M尿素的自氧化条件下，对结蛋白 - 波形蛋白混合物进行相同的分离，以获得两种蛋白质各自的交联碰撞复合物。在5M尿素中，氧化产物被鉴定为二聚体，其在物理上与未交联的同二聚体无法区分，这表明它们是以部分变性的面对面配对形式存在。来自完整中间丝的异二聚体与来自碰撞复合物的异二聚体相同。在2 - 巯基乙醇存在下，异二聚体不稳定并自发转化为同二聚体。去除尿素后，所有交联二聚体完全无法聚合成丝；然而，在2 - 巯基乙醇存在下，它们表现出正常的组装能力。氧化的同二聚体和异二聚体无法聚合，以及从细胞骨架中获得的交联二聚体产量相对较低，可能是由于二硫键形成给二聚体引入了空间应变。大量的交联异二聚体也可以从由同二聚体或四聚体形式的结蛋白和波形蛋白混合物重构的中间丝中分离出来。综上所述，这些结果表明，从细胞骨架中分离出的交联二聚体源于中间丝亚丝链之间的反应，而不是预先存在的二聚体内二硫键的形成，并且BHK - 21细胞的异型中间丝在很大程度上分别由同二聚体和四聚体形成，而不是由异二聚体形成。结蛋白和波形蛋白与ssDNA相互作用的差异能力也被用于区分同型和异型原丝，后者由每种蛋白质物种的一个同二聚体组成。这种区分可以基于各自蛋白质 - DNA复合物沉降行为的特征差异来进行。
