Figure 6. Influence of the proton net back flux capacity on the extent of the fast acidification process.

Comparison of the uncatalysed acidification (A) with a control assay (B) and experiments with retarded back flux (C and D). The samples were incubated under conditions as described in Fig. 5. A, a typical uncatalysed reaction. An uncatalysed reaction is achieved either by adding ETX (10 μM) to the ghost suspension or by gassing PBS to which BCECF (free acid) had been added (see Fig. 7A and B). B, a control experiment, which is the time course of acidification according to the standard protocol. C, a retarded back flux under conditions that inhibit the Na⁺-H⁺ antiporter (10 μM EIPA, incubated for 30 min prior to acidification). The arrow indicates the more pronounced plateau phase (kinetic data of acidification using a mono-exponential fit, see Table 1). D demonstrates that a temperature-sensitive transport mechanism is involved in the back flux of acidic equivalents. The temperature was lowered to 25 °C and caused a significant drop in acidification after the onset of CO₂, since the elimination of acidic equivalents is retarded (kinetic data of acidification using a mono-exponential fit, see Table 1).