2.4. Assays for mitochondrial O2 consumption and ATP production

Freshly isolated mitochondria were assayed for O₂ consumption rate (Jo) and ATP production rate (Jp). O₂ consumption was measured polarographically in a respiration chamber (Hansatech Instruments, Norfolk, UK) at 37 °C following general procedures we have previously described [13]. Mitochondrial respiration was fueled with the substrate combination pyruvate (1 mM)+malate (1 mM)+glutamate (10 mM) (PMG). Aliquots, typically 20 μl in volume, of mitochondrial suspension were added to 250 μl of respiration medium adapted from Wanders et al. [14], which contained (in mM) 100 KCl, 50 MOPS, 10 K2PO4, 10 MgCl2, 1 EGTA, and 0.2% BSA, pH 7.00 [13]. Next, the PMG substrate combination was added and State 2 Jo was followed (respiration due primarily to proton leak). The addition of ADP to give a final concentration of 0.67 mM stimulated state 3, (maximal) Jo. Phosphorylation of this ADP resulted in state 4 Jo [15], and the respiratory control ratio (RCR) was calculated as state 3 Jo/state 4 Jo. The ADP/O ratio was determined as previously described [15]. The State 3 (maximal) rate of ATP production was calculated as the product of state 3 Jo times the ADP/O (taking the 2:1 molecular to atomic oxygen stoichiometry into account).