FIGURE 7.
Complex I activity is compromised but is recovered by restoration of redox environment. Measurement of complex I activity (a) and mitochondrial ROS production (b) in the absence or presence of 10 mm dithiothreitol (DTT). n = 4, mean ± S.E., Student's t test. c, complex I activity can be recovered by restoration of redox environment or the addition of exogenous glutaredoxin-1 (Grx). Mitochondria were permeabilized and then incubated for 5 min in various combinations of reduced glutathione (GSH), oxidized glutathione (GSSG) in the absence or presence of 10 units Grx. Complex I activity was then measured. The Eh values were estimated according to the Nernst equation (see Equation 1). n = 5, mean ± S.E., 1-way ANOVA with a post hoc Tukey's test. * denotes p < 0.05. d, estimation of the glutathionylation state of complex I subunit NDUSF1 (∼75 kDa) by immunoblot. Glutathionylation state was preserved by treating mitochondria with 25 mm N-ethylmaleimide. To afford a proper electrophoretic estimation of NDUSF1 glutathionylation, NDUSF1 and PSSG levels were detected on the same gels. Samples were also electrophoresed under reducing conditions (+2% v/v βMetOH) as a control for PSSG immunodetection. Bands were quantified using ImageJ. n = 3, mean ± S.E., Student's t test. Mice were age 9–12 weeks.