Walker M S, DeMoss J A
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77030.
J Biol Chem. 1993 Apr 25;268(12):8391-3.
The regulation of specific gene expression by nitrate in Escherichia coli is mediated by the NarX/NarQ-NarL system. Based on sequence homologies with a family of two-component regulatory systems in bacteria, NarL has been identified as a putative response regulator while NarX and NarQ were proposed to be alternative membrane-associated sensors that activate NarL in the presence of nitrate. To investigate the interaction of NarX and NarL in vitro, both proteins were purified from overproducing strains. Purified NarX was rapidly labeled when incubated with [gamma-32P] ATP but not with [alpha-32P]ATP in a reaction that required Mg2+ but was unaffected by nitrate. Incubation of the labeled NarX with purified NarL resulted in the transient phosphorylation of NarL. Both the phosphorylation and dephosphorylation of NarL required Mg2+, and neither reaction was affected by the presence of nitrate. NarL-phosphate, stabilized by the addition of EDTA, ran as a monomer on gel filtration. Dephosphorylation of the isolated NarL-phosphate required the addition of both Mg2+ and the NarX protein. The relative stabilities of the phosphorylated forms of the two proteins at different pH values were consistent with the proposal that, in analogy to other related two-component regulatory systems, NarX and NarL were phosphorylated on specific histidine and aspartate residues, respectively.
大肠杆菌中硝酸盐对特定基因表达的调控是由NarX/NarQ-NarL系统介导的。基于与细菌中一类双组分调节系统的序列同源性，NarL被鉴定为一种假定的应答调节因子，而NarX和NarQ被认为是在硝酸盐存在时激活NarL的替代性膜相关传感器。为了在体外研究NarX和NarL的相互作用，两种蛋白质均从过量表达菌株中纯化得到。纯化的NarX在与[γ-32P]ATP孵育时会迅速被标记，但与[α-32P]ATP孵育时则不会，该反应需要Mg2+，但不受硝酸盐影响。将标记的NarX与纯化的NarL一起孵育会导致NarL的瞬时磷酸化。NarL的磷酸化和去磷酸化都需要Mg2+，且这两个反应均不受硝酸盐存在的影响。通过添加EDTA稳定的磷酸化NarL在凝胶过滤中以单体形式迁移。分离的磷酸化NarL的去磷酸化需要同时添加Mg2+和NarX蛋白。两种蛋白质的磷酸化形式在不同pH值下的相对稳定性与以下提议一致：与其他相关的双组分调节系统类似，NarX和NarL分别在特定的组氨酸和天冬氨酸残基上被磷酸化。
