FIGURE 6.

GSNO reduction by TGR and Trxs. A, the ability of TGR_WT and Grx-less TGR to reduce GSNO was tested using NADPH as an electron donor and following NADPH oxidation (A₃₄₀ decrease). For TGR_WT, different GSNO and enzyme concentrations were assayed as follows: 2 nm TGR_WT and 250 μm (light gray box), 500 μm (dark gray box), and 1 mm (black box) GSNO concentrations, and 40 nm TGR_WT and 500 μm GSNO (dark gray circle). For Grx-less TGR, 2 nm enzyme and 500 μm GSNO were used (light gray right triangle). A reaction mixture without enzyme and containing 500 μm GSNO was run for comparison (white diamond). The initial NADPH concentration was 100 μm in all cases. Note that as both reduced NADPH and GSNO contribute to A₃₄₀, initial absorbance values vary according to GSNO concentrations. A marginal GSNO reduction activity is observed only at high TGR_WT. B, TGR_WT at 2 nm (black box), and Grx-less TGR (2 nm) plus 10 μm cTrx (dark gray circle) or 10 μm mTrx (light gray circle) were compared for GSNO reduction at 500 μm GSNO and 100 μm NADPH. No significant activity was found for either Trx. C, the effect of GSNO addition on the GR activity of TGR_WT was studied by comparing the time courses for NADPH oxidation (A₃₄₀ decrease) by wild-type TGR (5 nm) with GSSG 100 μm in the absence (black box) or presence (dark gray right triangle) of 500 μm GSNO. The time course of TGR_WT (5 nm) at 1 mm GSSG is included as a control of an inhibited TGR (light gray box). The results indicated that at the conditions assayed TGR is not inhibited by GSNO. Note that the differences in initial absorbance values for the reaction mixtures containing different GSNO concentrations are due to the contribution of GSNO to A₃₄₀.