The activity of the mitochondrial respiratory chain complexes and respiratory rates was measured on cell homogenates, as described previously (62). The activity of the mitochondrial respiratory chain complexes was measured with cell homogenates in a cell buffer [250 mM saccharose, 20 mM tris(hydroxymethyl)aminomethane, 2 mM EGTA, and bovine serum albumin (BSA; 1 mg/ml) (pH 7.2)] at 37°C using a UV-SAFAS spectrophotometer (SAFAS). Cellular protein content was determined with the BCA (bicinchoninic assay) Kit (Pierce) using BSA as a standard. Complex I (NADH ubiquinone reductase, EC 1.6.5.3) activity was measured according to a procedure described elsewhere (63) and adapted using 2,6-dichloroindophenol (DCPIP) to avoid the inhibition of complex I activity by decylubiquinol (64). Cells were disrupted by two freezing-thawing cycles, washed, centrifuged for 1 min at 16,000g, and resuspended in cell buffer (50 μl per 106 cells). Complex II (succinate ubiquinone reductase) activity was measured according to James and colleagues (65). Specific enzymatic activities of complexes I and II were expressed in mIU (nanomoles of DCPIP per minute per milligram of protein). Complex IV (cytochrome c oxidase) activity was recorded according to a method that Rustin and colleagues (66) adapted in a 50 mM KH2PO4 buffer, using 15 μM reduced cytochrome c. Specific enzymatic activity was expressed in mIU (nanomoles of cytochrome c per minute per milligram of protein). CS activity was assayed by a standard procedure (67). Specific enzymatic activity was expressed in mIU [nanomoles of 5-5′-dithiobis(2-nitrobenzoic acid) per minute per milligram of protein].

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