Fig. 7.

Result of GSH and Grx2 on Ogdh-mediated ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ in liver mitochondria. (A) Effectiveness of alamethicin in permeabilizing mitochondria was tested by measuring malate dehydrogenase (Mdh) activity. Mitochondria were depleted of GSH by CDNB, permeabilized, and then re-supplemented with GSH (5 mM) or Grx2 (0.5 µM)+1 mM GSH. Reaction mixtures consisted of NAD⁺ (1 mM) and measurements were initiated following addition of malate. (B) CDNB depletes mitochondrial glutathione. Mitochondria were treated with or without CDNB (10 µM), permeabilized with alamethicin (40 µg/mL). Glutathione levels were estimated using Ellman's reagent. To ensure that GSH added to reaction mixtures could penetrate the matrix, permeabilized mitochondria pre-incubated in CDNB were re-supplemented with 5 mM GSH. (C and D) Exogenously added GSH amplifies ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ production by Ogdh which is reversed by glutaredoxin-2 (Grx2). Incubations were performed as described in A after which ${\mathrm{O}}_2^{\bullet -}$/H₂O₂ genesis and NADH production were examined. n=6, 1-way ANOVA with Tukey's post-hoc test.