Skip to main content
. 2014 Dec 15;111(52):18572–18577. doi: 10.1073/pnas.1417816112

Fig. 1.

Fig. 1.

Identification of Adh6 as the NADPH-dependent SNO-CoA reductase in yeast. (A) SNO-CoA–dependent NADPH consumption in yeast (S. cerevisiae). Extracts (800 µg/mL) were incubated with 100 μM SNO-CoA and 100 μM NADPH, and NADPH consumption (absorbance at 340 nm) was monitored continuously. (B) SNO-CoA–metabolizing activity in yeast extracts requires NADPH and not NADH. Extracts were incubated with 200 μM SNO-CoA and 100 μM NADPH or NADH and monitored continuously for 1 min. Data are presented as mean ± SD; n = 3. (C) SNO-CoA–mediated protein S-nitrosylation. Representative Coomassie-stained SDS/PAGE gel displaying SNO-proteins isolated by SNO-RAC following incubation of yeast lysate for 10 min with SNO-CoA (60 μM) alone or in combination with NADPH or NADH (100 μM). Ascorbate (Asc) was omitted from the SNO-RAC assay as a specificity control. Results are representative of three independent experiments. (D) Isolation and identification of SNO-CoA reductase. Representative Coomassie-stained SDS/PAGE gel corresponding to the five-step chromatographic purification scheme detailed in Table S1, which yielded from a crude extract (lane 1) 2,826-fold enrichment of NADPH-dependent SNO-CoA reductase activity identified by MS as Adh6 (lane 6).