ALTERATION OF NF-KAPPA-B P50 DNA-BINDING KINETICS BY S-NITROSYLATION

Nitric oxide (NO) regulates a wide variety of cellular functions, in p art, by formation of S-NO bonds at critical active site thiol groups w ithin proteins, including transcription factors. Previous studies have qualitatively demonstrated that S-nitrosothiol formation can alter tr anscription factor binding to the DNA recognition site. To more precis ely define the effect of S-nitrosylation on transcription factor bindi ng, the equilibrium binding constant was derived for S-nitrosylated NF -kappa B p50 (S-NO-p50) in a cell free system utilizing gel shift assa ys. Binding of NF-kappa B p50 subjected to the nitrosylation condition s in the absence of NaNO2 (C-p50-2) was not different from that of wil d 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 not ed 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. (C) 1997 Academic Press.