Mitochondrial complex activity assays

Complex I, II, and IV activities were measured in muscle tissue protein lysates using the Complex Enzyme Activity Microplate Assay Kit (Abcam) specific to each complex. Protein was isolated from the quadriceps of 1-year old animals. Briefly, minced tissue was incubated in 0.05% Trypsin-EDTA (Gibco) for 30 min on ice and agitated every few minutes. The tissue was then collected by centrifugation for 10 min at 4 °C, the supernatant discarded, and the muscle washed in cold mitochondrial isolation buffer (MIB; 200 mM mannitol, 68 mM sucrose, 10 mM HEPES pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.1% BSA) and centrifuged again. The muscle was resuspended in fresh MIB and then transferred to a glass dounce tube on ice. MIB was used to make up the remaining volume, and the tissue was homogenized with approximately 40 passes of the dounce pestle. Tissue homogenate was then transferred to a fresh Eppendorf tube, and detergent supplied with the Abcam kit was added. Samples were incubated for 30 min on ice, and lysate was collected by centrifugation at the appropriate speed and time for the specified assay kit. Protein concentration was determined by BCA assay (Pierce). Once lysate was obtained, the manufacturer’s protocol was followed to determine complex activity. Both MIB and the appropriate complex incubation buffer (Abcam kit) were used as individual negative controls for the assay, whereas bovine heart mitochondria isolate was used as a positive control.