The H+/ATP ratio is an important parameter for energy balance in cells and for the mechanism of coupling between proton transport and ATP synthesis. Rotational catalysis predicts that the H+/ATP coincides with the ratio of the c-subunits to the β-subunits, implying that a value of 4.7 is expected in the chloroplast ATPsynthase and a value of 3.3 is expected in the mitochondria and Escherichia coli enzyme. This ratio can be determined based on the energetics given by the chemiosmotic theory. The isolated enzymes were reconstituted into liposomes. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, so that the internal pH could be measured with a glass electrode. An acid-base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of ΔpH at constant Q = [ATP] / ([ADP] [Pi]). From the shift of the equilibrium ΔpH as a function of Q, the standard Gibbs free energy for phosphorylation and the H+/ATP ratios were determined.
Petersen J., Turina P., Graber P. (2008). The H+/ATP ratio of H+-ATPsynthases from chloroplasts, E. coli and mitochondria [10.1016/j.bbabio.2008.05.061].
The H+/ATP ratio of H+-ATPsynthases from chloroplasts, E. coli and mitochondria
TURINA, MARIA PAOLA;
2008
Abstract
The H+/ATP ratio is an important parameter for energy balance in cells and for the mechanism of coupling between proton transport and ATP synthesis. Rotational catalysis predicts that the H+/ATP coincides with the ratio of the c-subunits to the β-subunits, implying that a value of 4.7 is expected in the chloroplast ATPsynthase and a value of 3.3 is expected in the mitochondria and Escherichia coli enzyme. This ratio can be determined based on the energetics given by the chemiosmotic theory. The isolated enzymes were reconstituted into liposomes. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, so that the internal pH could be measured with a glass electrode. An acid-base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of ΔpH at constant Q = [ATP] / ([ADP] [Pi]). From the shift of the equilibrium ΔpH as a function of Q, the standard Gibbs free energy for phosphorylation and the H+/ATP ratios were determined.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
