METHODS

Care and training of animals. Male Carworth CFN rats weighing approximately 110 g at the start of the experiment were housed in individual cages and fed Wayne Lab Blox ad libitum. Half of the rats were allotted to a nontrained group, and the other half were assigned to a trained group which exercised 5 days/week for 12 weeks on the treadmill(Quinton Instrument Company, Seattle, Wash. 98 199) running schedule described by Holloszy (13). At the end of the training period, six of the untrained and five of the trained rats were run to exhaustion at 13 m/min, 8% grade, and 34 m/min, 8% grade, respectively. The exhausted animals were sacrificed by decapitation immediately after they were taken from the treadmill and their rested counterparts were killed at approximately the same time.

Isolation of mitochondria. Mitochondria were isolated by a modification of the method of Ernster and Nordenbrand (8). After decapitation, the quadriceps muscle group and heart were quickly removed, immersed in ice-cold Chappel-Perry media (8), and homogenized in an Omni mixer for 5 set at 16,000 rpm. Subsequently, the homogenate was transferred to a glass-Teflon Potter-Elvehjem homogenizer and homogenized further with five strokes of a pestle rotating at 1,200 rpm. The homogenate was centrifuged at 600 X g (maximum) for 10 min, the supernatant was saved, and the pellet resuspended in 5 ml Chappel-Perry media per gram of tissue. Following a second centrifugation at 600 X g for 10 min, the first and second supernatants were combined and spun at 15,000 X g for 10 min. After discarding the 15,000 X g supernatant, the mitochondrial pellet was resuspended in 10 ml of 0.15 M KC1 and centrifuged again at 15,000 X g. Finally, the mitochondrial pellet was suspended in 0.15 M KC1 to give approximately 10 mg mitochondrial protein per milliliter. Assay methods. Oxygen uptake was monitored in either an Oxygraph(Gilson Medical Electronics, Middleton, Wis.) with the Clark electrode or a YSI (Yellow Springs Instrument Co., Yellow Springs, Ohio) model-53 oxygen monitor. The reaction chambers of the oxygen electrodes were designed to contain 1.0 ml of reaction mixture. The final concentration of the components of the buffer system (pH 7.3) used for these studies was, in millimoles per liter: sucrose, 150; KCI, 15 (added with 0.1 ml of mitochondrial suspension), Tris-hydrochloride, 25; KzHP04, 6; EDTA, 0.5; and 0.3% bovine serum albumin. The substrates used