Fig. 5.

GSH alters electron flux in purified Ogdh resulting in amplified ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ production. (A) Representative traces showing the effect of different GSH concentrations (0–10 mM) on ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ and NADH production. Note that GSH≥5 mM completely abolishes NADH formation. Traces were utilized to calculate rates of ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ and NADH production (B). (C) Dose–response curves for the effect of GSH on Ogdh activity and ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ formation. (D) Impact of Ogdh inhibitor 3-methyl-2-oxopentanoic acid (KMV) on ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ and NADH production in the presence or absence of 5 mM GSH. (E) Autooxidation of AUR by different reaction mixture components. Calculated rates ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ production were compared to reactions containing Ogdh and Ogdh+5 mM GSH. Note that GSH (5 mM) and dithiol DTT (5 mM) induce a small change in the rate of AUR conversion to resorufin. (F) GSH has little effect on the rate of ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ production during reverse electron transfer from NADH. Reactions were initiated by the addition of NADH (5 µM) and then the reaction of AUR with H₂O₂ was measured. n=4, 1-way ANOVA with Tukey's post-hoc test.