We have recently shown that the H+/ATP ratio can significantly decrease during ATP hydrolysis by the ATPsynthase of Rb. capsulatus, when the concentration of either ADP or Pi is maintained at a low level. This same phenomenon has then been observed in isolated membranes of E. coli. We have now purified the ATPsynthase of E. coli and reconstituted it into liposomes, in order to verify whether the same behavior could be observed in the isolated enzyme. The ATP hydrolysis and proton pumping activity were measured under the same experimental conditions. The hydrolysis was measured either with the colorimetric pH indicator Phenol Red or with an ATP regenerating enzymatic assay, and the proton pumping was evaluated by a calibrated ACMA assay. The hydrolysis activity was inhibited by Pi with an apparent Kd of 400 μM, while the steady state ΔpH was stimulated up to 200 μM Pi and was only slightly inhibited up to 1000 μM Pi. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. We conclude that the isolated and reconstituted ATPsynthase of E. coli can vary its degree of coupling as a function of Pi and ADP.

Effect of Pi and ADP on the intrinsic uncoupling in the isolated and reconstituted ATPsynthase of E-coli

D'ALESSANDRO, MANUELA;TURINA, MARIA PAOLA;MELANDRI, BRUNO ANDREA
2008

Abstract

We have recently shown that the H+/ATP ratio can significantly decrease during ATP hydrolysis by the ATPsynthase of Rb. capsulatus, when the concentration of either ADP or Pi is maintained at a low level. This same phenomenon has then been observed in isolated membranes of E. coli. We have now purified the ATPsynthase of E. coli and reconstituted it into liposomes, in order to verify whether the same behavior could be observed in the isolated enzyme. The ATP hydrolysis and proton pumping activity were measured under the same experimental conditions. The hydrolysis was measured either with the colorimetric pH indicator Phenol Red or with an ATP regenerating enzymatic assay, and the proton pumping was evaluated by a calibrated ACMA assay. The hydrolysis activity was inhibited by Pi with an apparent Kd of 400 μM, while the steady state ΔpH was stimulated up to 200 μM Pi and was only slightly inhibited up to 1000 μM Pi. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. We conclude that the isolated and reconstituted ATPsynthase of E. coli can vary its degree of coupling as a function of Pi and ADP.
15th European Bioenergetic Conference 2008
14
15
D'Alessandro M.; Turina P.; Melandri B.A.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/123013
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