Novel application of S-nitrosoglutathione-sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphideformation

Site-specific S-glutathionylation is emerging as a novel mechanism by which S-nitrosoglutathione (GSNO) may modify functionally important protein thio ls. Here, we show that GSNO-Sepharose mimicks site-specific S-glutathionyla tion of the transcription factors c-Jun and p50 by free GSNO in vitro. Both c-Jun and p50 were found to bind to immobilized GSNO through the formation of a mixed disulphide, involving a conserved cysteine residue located in t he DNA-binding domains of these transcription factors. Furthermore, we show that c-jun, p50, glycogen phosphorylase b, glyceraldehyde-3-phosphate dehy drogenase, creatine kinase, glutaredoxin and caspase-3 can be precipitated from a mixture of purified thiol-containing proteins by the formation of a mixed-disulphide bond with GSNO Sepharose. With few exceptions, protein bin ding to this matrix correlated well with the susceptibility of the investig ated proteins to undergo GSNO- but not diamide-induced mixed-disulphide for mation in vitro. Finally, it is shown that covalent GSNO-Sepharose chromato graphy of HeLa cell nuclear extracts results in the enrichment of proteins which incorporate glutathione in response to GSNO treatment. As suggested b y DNA-binding assays, this group of nuclear proteins include the transcript ion factors activator protein-1, nuclear factor-kappa B and cAMP-response-e lement-binding protein. In conclusion, we introduce GSNO-Sepharose as a pro be for site-specific S-glutathionylation and as a novel and potentially use ful tool to isolate and identify proteins which are candidate targets for G SNO-induced mixed-disulphide formation.