Mixed skeletal muscle mitochondrial populations were isolated by a standard extraction protocol, involving potter homogenization, partial protease digestion and differential centrifugations. Briefly, pectoral muscle was cut up finely with sharp scissors in an ice-cold isolation buffer containing 100 mmol l−1 sucrose, 50 mmol l−1 KCl, 5 mmol l−1 EDTA, 50 mmol l−1 Tris-base, and pH 7.4 at 4°C. The minced tissue was homogenized with a Potter-Elvehjem homogenizer (five passages) and centrifuged at 800 g for 10 min. The supernatant containing the subsarcolemmal mitochondria was centrifuged at 1000 g for 10 min and the pellet containing the intermyofibrillar mitochondria was re-suspended in 10 ml of isolation buffer and treated with subtilisin (1 mg g−1 muscle wet weight) for 5 min in an ice bath. The mixture was diluted 1:2, homogenized (three passages) and then centrifuged at 1000 g for 10 min. Both resulting supernatants were filtered through cheesecloth and centrifuged at 8700 g for 10 min to pellet subsarcolemmal and intermyofibrillar mitochondria. The pellets were suspended in 10 ml of isolation medium, mixed together and centrifuged at 8700 g for 10 min. The resulting pellet was washed once by suspension in the isolation buffer and re-centrifuged at 8700 g for 10 min. All steps were performed at 4°C. The protein concentration of mitochondrial suspensions was determined by a Biuret method with bovine serum albumin (BSA) as the standard.