Ribonucleotide reductase of Escherichia coli. Cross-linking agents as probes of quaternary and quinary structure.
Ribonucleotide reductase of Escherichia coli. Cross-linking agents as probes of quaternary and quinary structure.
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大肠杆菌的核糖核苷酸还原酶。
DOI：
10.1111/j.1432-1033.1987.tb13512.x
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Reichard,P
中科院分区：
文献类型：
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作者：
Mathews,CK;Sjöberg,BM;Reichard,P
利用纯化的B1和B2蛋白和双功能交联剂，研究了大肠杆菌核糖核苷酸还原酶的四级结构。已知全酶是一种α2β2四聚体，由两种二聚体蛋白B1 （α2）和B2 （β2）组成。交联数据支持两个β亚基仅与两个α亚基中的一个密切相互作用的模型。一些涉及B2的相互作用通过截断的B2蛋白（β ' β '）定位在蛋白质的C端，这是B2的蛋白质水解裂解产物，其中每个β亚基缺失了30个羧基末端残基。其他相互作用表明，戊二氧还蛋白，而不是硫氧还蛋白，能够抑制一些交联反应。我们还通过将交联剂直接添加到整个细菌的悬浮液中，询问核糖核苷酸还原酶是否与细胞内的其他蛋白质密切相互作用。这些交联细菌提取物中的蛋白质经电泳分离，并用B1蛋白单克隆抗体进行探针检测。检测到高分子质量产物，支持该方法用于鉴定DNA前体生物合成酶之间的细胞内相互作用。
The quaternary structure of ribonucleotide reductase ofEscherichia coliwas investigated, with the use of purified B1 and B2 proteins and bifunctional cross‐linking agents. The holoenzyme is known to be an α2β2tetramer consisting of two dimeric proteins: B1 (α2) and B2 (β2). The cross‐linking data support a model in which both of the β subunits interact closely with only one of the two α subunits. Some of the interactions involving B2 were localized to the C terminus of the protein by use of truncated B2 protein (β‘β’), a proteolytic cleavage product of B2 in which the 30 carboxy‐terminal residues are missing from each of the β subunits. Other interactions were indicated by the ability of glutaredoxin, but not thioredoxin, to inhibit some of the cross‐linking reactions.We also asked whether ribonucleotide reductase interacts closely with other proteins inside the cell, by adding cross‐linkers directly to suspensions of whole bacteria. Proteins in extracts of these cross‐linked bacteria were resolved electrophoretically and probed with a monoclonal antibody to the B1 protein. High‐molecular‐mass products were detected, supporting the utility of this method for identifying intracellular interactions among enzymes of DNA precursor biosynthesis.