Cation/proton antiport systems in Escherichia coli.

The calcium/proton antiporter of Escherichia coli was characterized by measurement of the effect of divalent cations on the respiration-coupled quenching of the fluorescence of quinacrine or 9-aminoacridine in everted membrane vesicles. Energy-linked quenching of the fluorescence of aminoacridines is dependent on proton uptake into everted vesicles and provides an indirect assay for measurement of the transmembrane pH gradient. Fluorescence quenching was partially reversed by addition of the divalent cations Ca’+, Mn’+, Sr’+, or Ba’+. The antiporter exhibited sigmoidal kinetics when the initial rate of fluorescence change was measured as a function of cation concentration. For each substrate, the value of the Hill coefficient approached 2. The SO.~ values indicate that the order of affinity of the antiporter for substrates was Ca2+ = Mn2+ > Sr2+ > Ba2+. Neither M$+ nor La3+ were found to be substrates, but both inhibited the reversal of the quenching of quinacrine fluorescence produced by substrates of the antiporter, inhibiting by about 90% at 50 mM MgClz or 50 pM LaCl+ In the presence of lower concentrations of MgClz or LaCls, the Hill coefficients for Ca2+ and S? were reduced from values approaching 2 to values approaching 1. These data suggest an allosteric mechanism in which both homotropic and heterotropic effects occur. The calcium/proton antiporter exhibited an apparent pH optimum of about 8. Dissipation of the membrane potential with permeant anions inhibited the response of the pH gradient, as measured by the fluorescent assay, to divalent cations. This suggests that the calcium/proton antiporter operates by an electrogenic mechanism in which the stoichiometry of H+:Ca2+ > 2. Alternatively, the absolute requirement for a membrane potential may reflect a gating of the antiporter or a potential-dependent binding of substrate.