KINETICS OF ATP SYNTHESIS CATALYZED BY THE H+-ATPASE FROM CHLOROPLASTS (CF0F1) RECONSTITUTED INTO LIPOSOMES AND CORECONSTITUTED WITH BACTERIORHODOPSIN

The H+-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, DELTApH, and a transmembrane electric potential difference, DELTApsi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant DELTApH and constant DELTApsi as a function of temperature between 5-degrees-C and 45-degrees-C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant DELTApH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red) . The results can be described by Michaelis-Menten kinetics with the following parameters: V(max) = 0.5 s-1, K(m) = 8 muM for E(ox) and V(Max) = 2.0 s-1, K(m) = 8 muM for E(red).