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H + /ATP ratio of proton transport‐coupled ATP synthesis and hydrolysis catalysed by CF 0 F 1 —liposomes
Author(s) -
Turina Paola,
Samoray Dietrich,
Gräber Peter
Publication year - 2003
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/cdg073
Subject(s) - chemiosmosis , atp synthase , atp hydrolysis , gibbs free energy , phosphatidic acid , electrochemical gradient , biochemistry , biology , atpase , chromatography , chemistry , phospholipid , membrane , enzyme , physics , quantum mechanics
The H + /ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H + ‐translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pH in , could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid—base transition at different [ATP]/([ADP][P i ]) ratios as a function of ΔpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the ΔpH at equilibrium, ΔpH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][P i ]) ratio as a function of ΔpH (eq) and of the transmembrane electrochemical potential difference, Δμ̃ H+ (eq), resulted in H + /ATP ratios of 3.9 ± 0.2 at pH 8.45 and 4.0 ± 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 ± 2 kJ/mol at pH 8.45 and 36 ± 3 kJ/mol at pH 8.05.