Figure 8.

Effect of a second Δψ pulse on transport yield and transport kinetics at different IMV concentrations. All assays were conducted under standard transport conditions (Fig. 1 C), unless otherwise noted. (A) Effect of delayed addition of oxygenated buffer on transport yield. This anti-SufI immunoblot shows the mean amount of pre-SufI transported when the reaction volume was doubled with prewarmed (37°C) oxygenated buffer (35 μL; no NADH) at various times after reaction initiation in the presence of 8 mM NADH and 5 μM nigericin (n = 3). The control lane shows the amount of pre-SufI transported after 30 min in the absence of NADH. Note that the ordinate shows transport yield, i.e., the amount of precursor transported (in pmol) rather than the percentage of precursor transported relative to some control. [pre-SufI] = 250 nM; [IMV] = 2 (A ₂₈₀). (B) Generation of a second Δψ pulse at the indicated times (arrowheads) after the initial Δψ pulse. Time delays correspond to the points in panel A. Identical conditions as panel A. ΔF (100%) corresponds to the total fluorescence observed at the beginning of the experiment (before NADH addition). (C) Kinetics of pre-SufI translocation at different IMV concentrations, normalized to the 30-min time point. [pre-SufI] = 50 nM; [IMV] = 2 (black), 4 (red), and 6 (blue) (A ₂₈₀).