Figure 2.

Activation of H⁺ transport in UCP2 and UCP3 by CoQ₁₀. H⁺ influx into phospholipid vesicles reconstituted with digitonin-treated IB-UCP2 and IB-UCP3 was recorded. (A) Recordings of H⁺ uptake in reconstituted UCP liposomes in the presence of CoQ₁₀ (2 nmol) and absence of LA, with LA (125 μM) and without CoQ₁₀, with CoQ₁₀ and LA, and the inhibition with 20 μM ATP. (B) Evaluated H⁺ transport rates. H⁺ influx was measured as the change in external pH monitored by pyranine fluorescence at λ_ex = 467 nm and λ_em = 510 nm in the presence of 125 μM LA. In all experiments (A and B) a 50-μl portion of vesicles containing 1.3 μg protein and 420 μg phospholipid was added to 0.5 mM Hepes buffer (pH 7.3) containing 1 μM pyranine, 0.5 mM EDTA, and 280 mM sucrose to a final volume of 330 μl at pH 6.8 and 10°C. H₂SO₄ was added in steps of 10 nmol H⁺ to adjust the pH to 6.8, and valinomycin (Val) was added to a concentration of 2.5 μM. Uncoupling by 1 μM carbonylcyanide m-chlorophenylhydrazone determined the capacity of H⁺ uptake of the vesicles. Results are presented as the initial H⁺ transport rate (V: μmol/min mg protein) divided by the capacity of the vesicles (C: μmol/ml).