Figure 3.

Distribution of incorporated ¹⁴C from SDS/PAGE analysis of [¹⁴C]DCCD-treated aN214C/cM65C-membranes. A membrane sample (1 mg protein/ml) was incubated for 2 h with 10 μM [¹⁴C]DCCD, resulting in 72% inhibition of ATPase activity. Aliquots were diluted and exposed to different conditions for 3 min, then incubated alone or with 50 μM I₂ for 10 min. Each aliquot then was denatured in the presence of 5 mM NEM and analyzed by nonreducing SDS/PAGE. The dried gel was exposed to a storage phosphor screen for ≈6 days. Shown are the profiles of radioactivity in sample lanes after scanning the phosphor screen. Locations of F_OF₁ subunits are noted. (A) Nonoxidized (black) and oxidized (green) controls. The peak at the migration front (labeled DCCD), represents [¹⁴C]DCCD that hydrolyzed or reacted with a buffer component. (B) Comparison of aliquots exposed to 4 mM ADP/P_i (blue) or 4 mM ATP (red) before oxidation. The offset, partial traces show the radiolabel in the a–c band for a sample treated with 100 μM [¹⁴C]DCCD for 0.5 h before the nucleotide incubation and oxidation steps noted above. The scale for the offset traces is about 3-fold greater than that shown on the y axis.