Quantitative evaluation of the intrinsic uncoupling modulated by ADP and Pi in the reconstituted ATP synthase of Escherichia coli

AbstractThe ATP synthase from Escherichia coli was isolated and reconstituted into liposomes. The ATP hydrolysis by these proteoliposomes was coupled to proton pumping, and the ensuing inner volume acidification was measured by the fluorescent probe 9-amino-6-chloro-2-methoxyacridine (ACMA). The ACMA response was calibrated by acid–base transitions, and converted into internal pH values. The rates of internal acidification and of ATP hydrolysis were measured in parallel, as a function of Pi or ADP concentration. Increasing Pi monotonically inhibited the hydrolysis rate with a half-maximal effect at 510μM, whereas it stimulated the acidification rate up to 100–200μM, inhibiting it only at higher concentrations. The ADP concentration in the assay, due both to contaminant ADP in ATP and to the hydrolysis reaction, was progressively decreased by means of increasing pyruvate kinase activities. Decreasing ADP stimulated the hydrolysis rate, whereas it inhibited the internal acidification rate. The quantitative analysis showed that the relative number of translocated protons per hydrolyzed ATP, i.e. the relative coupling ratio, depended on the concentrations of Pi and ADP with apparent Kd values of 220μM and 27nM respectively. At the smallest ADP concentrations reached, and in the absence of Pi, the coupling ratio dropped down to 15% relative to the value observed at the highest ADP and Pi concentrations tested. In addition, the data indicate the presence of two ADP and Pi binding sites, of which only the highest affinity one is related to changes in the coupling ratio