2.8. Mitochondrial respiration.

High-resolution respirometry (O₂ consumption) were conducted in 2 mL of respiration medium (Buffer Z) using the Oroboros Oxygraph-2k (Oroboros Instruments, Corp., Innsbruck, Austria) with stirring at 750 rpm at 37°C. Buffer Z contained 20 mM Cr to saturate mtCK and promote phosphate shuttling through mtCK or was kept void of Cr to prevent the activation of mtCK [³⁴]. All experiments were conducted in the presence of 5 μM blebbistatin (BLEB) in the assay media to prevent spontaneous contraction of PmFB, which has been shown to occur in response to ADP at 37°C that alters respiration rates [³⁴]. Complex I-supported respiration was stimulated using 5mM pyruvate and 2mM malate (NADH) followed by a titration of ADP concentrations from physiological ranges (25 μM, 100 μM; [³⁵]) to high submaximal (500 μM) and saturating to stimulate maximal coupled respiration (5000 μM in the presence of creatine and 7000 μM in the absence of creatine). 10mM glutamate (further NADH generation) was added at the end of the ADP titration. Cytochrome c was then added to test mitochondrial outer membrane integrity. Experiments with low ADP-stimulated respiration (bundles that did not respond to ADP) with high cytochrome c responses (>15% increase in respiration) were removed from analysis (13 of 370 bundles). Last, 20mM Succinate (FADH₂) was added to stimulate complex-II supported respiration. These protocols were designed to understand the regulation of respiration coupled to oxidative phosphorylation of ADP to ATP (Adenosine triphosphate).