Differential effects of nitric oxide-mediated S-nitrosylation on p50 and c-jun DNA binding.

BACKGROUND: Nitric oxide (NO) regulates a variety of cellular functions. One mechanism by which NO may exert its influence is through formation of S-nitrosothiols at critical thiol residues in protein-active sites, including those of nuclear protein transcription factors. METHODS: NF-kappa B p50 and AP-1 c-jun were S-nitrosylated in the presence of acidic NaNO2. Wild-type protein and protein subjected to nitrosylating conditions in the absence of NaNO2 served as controls. Confirmatory evidence for S-nitrosothiol bond formation was obtained by ultraviolet-visible spectrophotometry with the absorption maximum for S-NO bonds at approximately 320 to 360 nm. With consensus oligonucleotide probes, gel-shift analysis was used to examine DNA binding characteristics. RESULTS: In the case of NF-kappa B p50, S-nitrosylation resulted in significantly decreased DNA binding. In contrast, S-nitrosylation did not alter c-jun DNA binding. The S-nitrosylating conditions themselves did not alter p50 or c-jun DNA binding. Quantitative analysis was performed according to the Scatchard plot technique to generate the respective dissociation constants. S-nitrosylated p50 was associated with a fourfold greater dissociation constant than that of the wild-type p50. CONCLUSIONS: S-nitrosylation of transcription factors may be one mechanism by which NO may selectively regulate gene transcription.

Duke Scholars

Author Rebecca Ann Schroeder Anesthesiology, VA Anesthesiology Service

Author Paul C. Kuo Surgery