DelaTorre A, Schroeder R A, Kuo P C
Department of Surgery, University of Maryland Medical Center, Baltimore 21201, USA.
Biochem Biophys Res Commun. 1997 Sep 29;238(3):703-6. doi: 10.1006/bbrc.1997.7279.
Nitric oxide (NO) regulates a wide variety of cellular functions, in part, by formation of S-NO bonds at critical active site thiol groups within proteins, including transcription factors. Previous studies have qualitatively demonstrated that S-nitrosothiol formation can alter transcription factor binding to the DNA recognition site. To more precisely define the effect of S-nitrosylation on transcription factor binding, the equilibrium binding constant was derived for S-nitrosylated NF-kappa B p50 (S-NO-p50) in a cell free system utilizing gel shift assays. Binding of NF-kappa B p50 subjected to the nitrosylation conditions in the absence of NaNO2 (C-p50-2) was not different from that of wild type NF-kappa B (C-p50-1). The extent of S-NO-p50 binding to its DNA target sequence was significantly decreased in comparison to that noted with C-p50-1 and C-p50-2. The binding constant was derived for each of the NF-kappa B variants: C-p50-1 = 1.01 x 10(10) M(-1); C-p50-2 = 0.92 x 10(10) M(-1); and S-NO-p50 = 0.28 x 10(10) M(-1). These data indicate that S-nitrosylation of p50 decreases its affinity for the target DNA sequence by four-fold.
一氧化氮（NO）部分通过在包括转录因子在内的蛋白质关键活性位点硫醇基团处形成S-NO键来调节多种细胞功能。先前的研究已定性证明S-亚硝基硫醇的形成可改变转录因子与DNA识别位点的结合。为了更精确地定义S-亚硝基化对转录因子结合的影响，在无细胞系统中利用凝胶迁移试验得出了S-亚硝基化核因子-κB p50（S-NO-p50）的平衡结合常数。在不存在亚硝酸钠的情况下，处于亚硝基化条件下的核因子-κB p50（C-p50-2）的结合与野生型核因子-κB（C-p50-1）的结合没有差异。与C-p50-1和C-p50-2相比，S-NO-p50与其DNA靶序列的结合程度显著降低。得出了每个核因子-κB变体的结合常数：C-p50-1 = 1.01×10¹⁰ M⁻¹；C-p50-2 = 0.92×10¹⁰ M⁻¹；以及S-NO-p50 = 0.28×10¹⁰ M⁻¹。这些数据表明p50的S-亚硝基化使其对靶DNA序列的亲和力降低了四倍。
