Quantitative evaluation of the intrinsic uncoupling modulated by ADP and P(i) in the reconstituted ATP synthase of Escherichia coli.
The 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 P(i) or ADP concentration. Increasing P(i) 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 P(i) and ADP with apparent K(d) values of 220μM and 27nM respectively. At the smallest ADP concentrations reached, and in the absence of P(i), the coupling ratio dropped down to 15% relative to the value observed at the highest ADP and P(i) concentrations tested. In addition, the data indicate the presence of two ADP and P(i) binding sites, of which only the highest affinity one is related to changes in the coupling ratio.
D'Alessandro
Manuela
M
Department of Biology, Laboratory of Biochemistry and Biophysics, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.
Turina
Paola
P
Melandri
B Andrea
BA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002736:Chloroplasts / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism
D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D006180:Proton-Translocating ATPases