The reduction of dithiobis(2-nitrobenzoate) by rat liver mitochondria.

5,5'-Dithiobis(2-nitrobenzoate) (DTNB) is reduced in mitochondrial suspensions to 5-mercapto-2-nitrobenzoate (MNB) by 3-hydroxybutyrate and isocitrate. Although most of the MNB produced is found in the suspension medium, there is also some within the particles. The amount of MNB found in these fraction varies with the DTNB concentration used and is much lower if mitochondrial glutathione (GSH) is depleted with 1-chloro-2,4-dinitrobenzene. If hydroxybutyrate is present, the reduction of DTNB is increased by ATP and oligomycin. The pellet contains only a little MNB and GSH but these are considerably elevated by antimycin and rotenone as well as by ATP and oligomycin. If isocitrate is present, the reduction of DTNB is greatly stimulated by valinomycin, triethyltin and, to a lesser extent, oligomycin. MNB in the pellet falls and GSH concentrations are unchanged. The results suggest that with hydroxybutyrate (an NAD reducing substrate), the rate of reduction of DTNB is limited by the rate of regeneration of GSH while with isocitrate (an NADP reducing substrate) it is limited by the rate of export of MNB from the matrix.