2.5. Isolation of Mitochondria

Mitochondria were isolated from bovine or rat liver by a process of differential centrifugation at 4 °C [10]. Briefly, 5–6 g of tissue were minced and homogenized in STE (250 mM sucrose, 1 mM Tris-HCI, and 1 mM EDTA (ethylenediaminetetraacetic acid, pH 7.4)), 20% w/v, and centrifuged at 600× g for 10 min. The supernatant was collected and then centrifuged for 10 min at 8000× g. The resulting mitochondrial pellet was re-suspended and washed twice by centrifugation for 10 min at 8000× g in STE, followed by an additional wash in ST (250 mM sucrose and 1 mM Tris-HC1 (pH 7.4)). The final pellet was re-suspended in ST and the protein concentration of the mitochondrial suspension was determined by the method of Lowry [11].