Figure 5. Effects of rotenone, piericidin A, FCCP and nigericin on superoxide/hydrogen peroxide production during reverse electron transport (site IQr) and forward electron transport (site IQf).
Plate-reader assays of superoxide/hydrogen peroxide production by rat skeletal muscle mitochondria (0.2 mg/ml; 0.01 mg/well) oxidising glycerol-3-phosphate alone (reverse electron transport, site IQr) or in the presence of 50 µM glutamate plus malate (forward electron transport, site IQf), in the presence of 2 µM atpenin A5 to prevent electron backflow into site IIF and 10 µM S3QEL3 to suppress site IIIQo. Effects of (A) rotenone; (B) Piericidin A; (C) FCCP; (D) Nigericin, each added at the start of the experiment. Residual rates were not subtracted because they presumably include contributions from sites GQ and IF (and, with glutamate plus malate added, site OF) that alter during the titrations. Curves were fit using the non-linear curve function (variable slope; four parameter logistic) of Prism 7. Values are means ± SEM (n = 3 independent experiments each the mean of 3 technical replicates).