FIGURE 2.

Na⁺/H⁺ antiporter activity in everted membrane vesicles of NhaA mutants. Everted membrane vesicles were prepared from EP432 cells expressing the indicated NhaA mutants from pCL-HAH4 and grown in LBK (pH 7). The Na⁺/H⁺ antiporter activity was determined at the indicated pH values using acridine orange fluorescence to monitor ΔpH in the presence of 10 mm NaCl as described under “Experimental Procedures.” A, the data of typical experiments with CL-NhaA and mutant H253C are shown. At the onset of the reaction, ATP or d-lactate (2 mm) was added (↓), and the fluorescent quenching (Q) was recorded until a steady state level of ΔpH (100% quenching) was reached. NaCl (10 mm) was then added (↑), and the new steady state of fluorescence obtained (dequenching) after the addition was monitored. B, the percentage of dequenching as observed following the addition of 10 mm NaCl is shown versus pH. The results obtained with E252C (16) are also shown. C, experiments as in B with mutants F267C, F344C, and F267C/F344C. All experiments were repeated at least three times with practically identical results.