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. Author manuscript; available in PMC: 2010 Aug 30.
Published in final edited form as: Chem Biol Interact. 2008 Nov 18;178(1-3):250–258. doi: 10.1016/j.cbi.2008.11.007

Fig. 2. Inactivation of S-nitrosylated AR by reduced glutathione.

Fig. 2

(A) Reduced AR was incubated with 0.5 mM DEANO for 1h at 25°C, desalted and then incubated with 2 mM GSH in 0.1 M potassium phosphate (pH 7.0) for 1h at 25°C. Aliquots were withdrawn from the reaction mixture and the enzyme activity was measured as described under Materials and Methods in the presence and absence of 1 μM sorbinil, as indicated. Values are expressed as mean ± S.D, (n = 3) #P< 0.05 versus non-treated protein and *P <0.05 versus DEANO-treated protein. (B) Electrospray mass spectra of (i) reduced WT AR treated with DEANO showing molecular ions for unmodified AR (37,885 Da) and ARS-NO (37,918 Da); (ii) DEANO-modified AR incubated with 2 mM GSH for 1 h showing both, native (37,885 Da) and glutathiolated (+ 306 Da) AR (38,190 Da); (iii) reduced WT AR incubated with GSH showing only the native protein (37,885 Da). (C) Western blot analysis of DEANO-treated AR incubated with GSH. WT AR was treated with DEANO, desalted and treated with 2 mM GSH for the indicated time periods. The Western blots were developed using monoclonal anti-PSSG antibody. Lower panel shows group data for the intensity of the anti-PSSG immunopositive bands, normalized to AR. Data are presented as mean ± SD. #P < 0.005 versus DEANO-treated protein incubated with GSH for 0 min (n = 3).