Mitochondrial isolation and measurements of ETC complex activity

Intact brain mitochondria were isolated from post-mortem human or mouse brain tissue using differential centrifugation with digitonin treatment⁷³. Brain tissue was immersed into ice-cold isolation medium (225 mM mannitol, 75 mM sucrose, 20 mM HEPES-Tris, 1 mM EGTA, pH 7.4), supplemented with 1 mg/ml BSA. Tissue was homogenized with 40 strokes by pestle “B” (tight) of a Dounce homogenizer in 10 ml of isolation medium, diluted two-fold, and transferred into centrifuge tubes. The homogenate was centrifuged at 5900 × g for 4 min in a refrigerated (4 °C) Beckman centrifuge. The supernatant was centrifuged at 12,000 × g for 10 min and pellets were resuspended in the same buffer, and 0.02% digitonin was added. The suspension was homogenized briefly with five strokes in a loosely fitted Potter homogenizer and centrifuged again at 12,000 × g for 10 min, then gently resuspended in the isolation buffer without BSA and washed once by centrifuging at 12,000 × g for 10 min. The final mitochondrial pellet was resuspended in 0.1 ml of washing buffer and stored on ice. The respiratory activities were measured in Oroboros high-resolution respirometer as previously described⁷⁴.

The activity of the ETC complexes was measured spectrophotometrically using a plate reader (SpectraMax M5, Molecular Devices, USA) in 0.2 ml of standard respiration buffer composed of 125 mM sucrose, 25 mM Tris-HCl (pH = 7.5), 0.01 mM EGTA, and 20 mM KCl at 25 °C. NADH-dependent activity of complex I was assayed as oxidation of 0.15 mM NADH at 340 nm (ε340 nm = 6.22 mM⁻¹cm⁻¹) in the assay buffer supplemented with 10 µM cytochrome c, 40 µg/ml alamethicin, 1 mM MgCl₂ (NADH media). NADH:Q reductase was measured in NADH media containing 2 mg/ml BSA, 60 µM decylubiquinone, 1 mM cyanide and 5–15 µg protein per well. Only the rotenone (1 µM)-sensitive part of the activity was used for calculations. NADH:HAR reductase was assayed in NADH media containing 1 mM HAR and 2–5 µg protein per well. Complex II succinate:DCIP reductase activity was recorded at 600 nm (ε600 nm = 21 mM⁻¹cm⁻¹) in the KCl assay buffer (125 mM KCl, 20 mM HEPES-Tris, 0.02 mM EGTA, pH 7.6) containing 15 mM succinate, 40 µM decylubiquinone, 0.1 mM DCIP, 1 mM KCN, and 5–10 µg protein per well. Complex IV ferrocytochrome c oxidase activity was measured as oxidation of 50 µM ferrocytochrome c at 550 nm (ε550 nm = 21.5 L ∙ mM⁻¹cm⁻¹) in KCl assay buffer supplemented with 0.025% dodecylmaltoside and 1–3 µg protein per well. To assess the effect of CP2 on the activity, we pre-incubated 20–40 µg/ml mitochondria with various concentrations of CP2 for 10 min at 25 °C in the absence of substrates and then measured the residual activity as described above.