Traub P, Nelson W J, Kühn S, Vorgias C E
J Biol Chem. 1983 Feb 10;258(3):1456-66.
The binding of the intermediate filament protein vimentin to a variety of naturally occurring RNAs and DNAs was studied. The relative capacities of the various nucleic acids to associate with pure [3H]vimentin were determined in competition experiments with 28 S rRNA from Ehrlich ascites tumor cells. The reaction products were analyzed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Under these ionic conditions, vimentin reacted preferentially with single-stranded nucleic acids, particularly with those of high (G + C) content. The vimentin binding potentials of single-stranded RNAs and DNAs were largely comparable. However, when the concentrations of mono- and divalent cations were raised to physiological and higher values, only single-stranded DNA retained its vimentin binding capacity. With increasing KCl concentrations at 0 to 1 mM Mg2+, increasing amounts of vimentin were detected in complexes which sedimented considerably faster than the bulk of the DNA, suggesting cooperative binding of vimentin. The salt optimum of this cooperativity was at 200 mM KCl. Thus, the capability of vimentin to discriminate between single-stranded RNA and DNA under physiological ionic conditions points to specificity of the interaction of vimentin with nucleic acids.
研究了中间丝蛋白波形蛋白与多种天然存在的RNA和DNA的结合情况。在与艾氏腹水瘤细胞的28S rRNA的竞争实验中,测定了各种核酸与纯[3H]波形蛋白结合的相对能力。反应产物在低离子强度和存在EDTA的条件下通过蔗糖梯度离心进行分析。在这些离子条件下,波形蛋白优先与单链核酸反应,特别是与那些(G + C)含量高的核酸反应。单链RNA和DNA的波形蛋白结合潜力在很大程度上相当。然而,当单价和二价阳离子的浓度升高到生理值及更高值时,只有单链DNA保留其波形蛋白结合能力。在0至1 mM Mg2+存在下,随着KCl浓度的增加,在比大部分DNA沉降快得多的复合物中检测到越来越多的波形蛋白,这表明波形蛋白存在协同结合。这种协同作用的盐最佳浓度为200 mM KCl。因此,波形蛋白在生理离子条件下区分单链RNA和DNA的能力表明波形蛋白与核酸相互作用具有特异性。