IDENTIFICATION OF PROTEINS AT THE SUBUNIT INTERFACE OF THE ESCHERICHIA-COLI RIBOSOME BY CROSS-LINKING WITH DIMETHYL 3,3'-DITHIOBIS(PROPIONIMIDATE)
IDENTIFICATION OF PROTEINS AT THE SUBUNIT INTERFACE OF THE ESCHERICHIA-COLI RIBOSOME BY CROSS-LINKING WITH DIMETHYL 3,3'-DITHIOBIS(PROPIONIMIDATE)
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DOI：
10.1021/bi00513a021
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
TRAUT, RR
中科院分区：
文献类型：
--
作者：
COVER, JA;LAMBERT, JM;TRAUT, RR
E.大肠杆菌用二甲基3，3“-二硫代双（丙酰亚胺）处理。在40%的赖氨酸ε-氨基被修饰，约50%的核糖体成为抵抗解离成30 S和50 S亚基时，在含有低镁浓度的蔗糖梯度的还原剂的情况下进行分析。在还原剂存在下发生解离，表明双功能试剂与来自两个亚基的蛋白质反应。通过使用排除二硫键交换的条件从纯化的交联70 S核糖体中提取蛋白质。二硫键连接的蛋白质复合物和非交联的蛋白质通过在pH 5.5的聚丙烯酰胺/尿素凝胶中电泳进行分级。通过二维对角聚丙烯酰胺/十二烷基硫酸钠凝胶电泳分析来自尿素凝胶连续切片的蛋白质。衍生自交联二聚体的单体蛋白质出现在非交联蛋白质的对角线下方，因为第二次电泳而不是第一次电泳是在还原条件下进行的，以切割交联物质。每个二聚体中的组成蛋白的最终鉴定是通过交联蛋白的放射性碘标记，然后在非放射性标记物70 S蛋白的存在下进行二维聚丙烯酰胺/尿素凝胶电泳。鉴定了11个交联蛋白质二聚体，其中包含来自2个核糖体亚基中的每一个的1个蛋白质。这些交联对中的蛋白质显然位于2个亚基之间的接触区域，即，在子单元接口处。
The 70S ribosomes of E. coli were treated with dimethyl 3,3''-dithiobis(propionimidate). Under conditions where 40% of the lysine .epsilon.-amino groups became modified, about 50% of the ribosomes became resistant to dissociation into 30S and 50S subunits when analyzed in the absence of reducing agents on sucrose gradients containing low Mg concentrations. Dissociation took place in the presence of reducing agents, indicating that the bifunctional reagent had reacted with proteins from both subunits. Proteins were extracted from purified cross-linked 70S ribosomes by using conditions to preclude disulfide interchange. Disulfide-linked protein complexes and non-cross-linked proteins were fractionated by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gel were analyzed by 2-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Monomeric proteins derived from cross-linked dimers appeared below the diagonal of non-cross-linked proteins since the 2nd electrophoresis but not the first is run under reducing conditions to cleave the cross-linked species. Final identification of the constituent proteins in each dimer was made by radioiodination of the cross-linked proteins, followed by 2-dimensional polyacrylamide/urea gel electrophoresis in the presence of nonradioactive marker 70S protein. The identification of 11 cross-linked protein dimers which contained 1 protein from each of the 2 ribosomal subunits is described. The proteins in these cross-linked pairs are apparently located in the regions of contact between the 2 subunits, i.e., at the subunit interface.