Figure 12.

Carboxyfluorescein efflux from POPC/POPG mixtures induced by direct addition of cecropin A (top panels) to POPC/POPG (A) 50:50, (C) 70:30, and (E) 80:20. In each top panel, the shaded curves correspond to lipid concentrations of 25, 50, 100, and 200 μM lipid (plus 400 μM in E). The fastest curve is always that for 25 μM lipid and the slowest is that for 200 μM lipid (400 μM in E). The peptide concentration is always 1 μM. The bottom panels show the CF efflux reverse experiments for the same vesicle compositions (shaded curves): POPC/POPG (B) 50:50, (D) 70:30, and (F) 80:20. Here, 60 μM donor vesicles containing no CF were incubated with 2 μM peptide for the amount of time required to reach a plateau in the fraction of dye released in the regular CF efflux experiments (top). This corresponded to incubation times of 30, 40, and 60 min for POPC/POPG 50:50, 70:30, and 80:20, respectively. After incubation with peptide, the empty donor vesicles were mixed with an equal volume of 40 μM, CF-containing acceptor vesicles, resulting in a final 50 μM total lipid and 1 μM peptide concentration. The maximum possible fluorescence levels were determined by adding the detergent Triton X-100 to the vesicle suspension at a concentration of 1%, which dissolves the vesicles. In all panels, the curves shown are averages of approximately four independent traces recorded on the same vesicle preparation. In addition, the data was reproduced at least twice in independent vesicle preparations. In the top three panels, the thin solid lines are the fits of the model of Fig. 3, and correspond to the integrated differential Eqs. 13–17. The values obtained for the only variable parameter in these fits, β = k₁/k₂, are listed in Table 1. The fits also used the values of k_on and k_off independently obtained from the binding and dissociation kinetics, and the fixed parameters k_eflx = 100 M⁻¹ s⁻¹ and k₁ = 0.01 s⁻¹. In the bottom three panels, the dashed line is calculated using the exact values obtained from the corresponding fits in the top panels (Table 1). Allowing for a slight adjustment of β to fit the reverse experiment curves, the solid line is obtained, which corresponds to the values β = 1.4 × 10⁻³ for (B) 50:50, (D) 1.0 × 10⁻³ for 70:30, and (F) 0.73 × 10⁻³ for 80:20.