For all respiration experiments, mitochondria were incubated in RM at 37°C in the presence of sodium and in the absence of exogenous substrates or energy phosphates in order to deplete endogenous Ca2+ from the mitochondrial matrix (5 (link), 6 (link), 9 (link)). After the depletion step, 10 mM Pi and 0.13 mM ADP were added, followed by glutamate (G, 10 mM) and malate (M, 1 mM) ± Ca2+. Addition of fuel (G+M) initiated a submaximal increase in Jo followed by a subsequent transition to State 4 Jo. For experiments designed to measure maximal Jo, State 3 was then elicited with a bolus of 1.3 mM ADP which resulted in consumption of all the oxygen in the chamber providing the fully reduced state (anoxia). Free Ca2+ concentrations were made using the calculator programs of Fabiato and Fabiato (26 (link)) translated to Labview VIs (National Instruments Corp, Austin, TX) by Reitz and Pollack (27 (link)).
Steady-state, intermediate Jo was attained using a progressive creatine kinase (CK) energetic clamp (28 (link)), we have recently modified (29 (link)). Briefly, by utilizing a large total creatine pool, excess CK, a known [ATP], and the CK equilibrium constant (KCK, 150 (30 (link))), the extramitochondrial ATP/ADP ratio, and thus, free energy of ATP hydrolysis (ΔGATPe) can be calculated from the added phosphocreatine (PCr)/creatine (Cr) ratio:
where ΔGATP° is the standard ΔGATP (-7.592 kcal/mol), R is the gas constant (1.987 cal·K-1·mol-1), and T is temperature (310°K). Mitochondria oxidizing G+M at State 4 were given 2.5 mM PCr, 5 mM Cr, 5 mM ATP, and 75 U/ml CK, resulting in a Jo of ∼2/3 of State 3. Subsequent additions of PCr (to 3.75, 5, 7.5, and 10 mM) were made to slow Jo to ∼1/3 of State 3.
Additional experiments with pyruvate were conducted similar to those from Messer et al. (28 (link)). Briefly, mitochondria in the presence of saturating M were exposed to the CK clamp as described above with a PCr/Cr ratio of 0.25. Pyruvate was then titrated in stepwise at 10, 25, 50, 100, and 500 μM to increase respiration. The novel aspect of this experiment was that upon reaching a steady state with 500 μM pyruvate, stepwise additions of PCr were then made to slow Jo. Thus, in a single experiment, Jo was changed by both “Push” (pyruvate titration) and “Pull” (CK clamp) mechanisms.
Steady-state, intermediate Jo was attained using a progressive creatine kinase (CK) energetic clamp (28 (link)), we have recently modified (29 (link)). Briefly, by utilizing a large total creatine pool, excess CK, a known [ATP], and the CK equilibrium constant (KCK, 150 (30 (link))), the extramitochondrial ATP/ADP ratio, and thus, free energy of ATP hydrolysis (ΔGATPe) can be calculated from the added phosphocreatine (PCr)/creatine (Cr) ratio:
where ΔGATP° is the standard ΔGATP (-7.592 kcal/mol), R is the gas constant (1.987 cal·K-1·mol-1), and T is temperature (310°K). Mitochondria oxidizing G+M at State 4 were given 2.5 mM PCr, 5 mM Cr, 5 mM ATP, and 75 U/ml CK, resulting in a Jo of ∼2/3 of State 3. Subsequent additions of PCr (to 3.75, 5, 7.5, and 10 mM) were made to slow Jo to ∼1/3 of State 3.
Additional experiments with pyruvate were conducted similar to those from Messer et al. (28 (link)). Briefly, mitochondria in the presence of saturating M were exposed to the CK clamp as described above with a PCr/Cr ratio of 0.25. Pyruvate was then titrated in stepwise at 10, 25, 50, 100, and 500 μM to increase respiration. The novel aspect of this experiment was that upon reaching a steady state with 500 μM pyruvate, stepwise additions of PCr were then made to slow Jo. Thus, in a single experiment, Jo was changed by both “Push” (pyruvate titration) and “Pull” (CK clamp) mechanisms.
