title: Alteration of NF-kappa B p50 DNA binding kinetics by S-nitrosylation.
abstract: 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.
authors.affiliation: Department of Surgery, University of Maryland Medical Center, Baltimore 21201, USA.
authors.forename: A
authors.initials: A
authors.lastname: DelaTorre
authors.forename: R A
authors.initials: RA
authors.lastname: Schroeder
authors.forename: P C
authors.initials: PC
authors.lastname: Kuo
mesh_terms: D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D052002:NF-kappa B p50 Subunit; D009569:Nitric Oxide / Q000378:metabolism* / Q000502:physiology
chemical_list: D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009569:Nitric Oxide; D004247:DNA