ADP binding and ATP synthesis by reconstituted H+-ATPase from chloroplasts

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Abstract
The H+-ATPase from chloroplasts, CF0F1, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pHin = 5.0, pHout = 8.5) and a K+/valinomycin diffusion potential, Δφ (K+ in = 0.6 mM, K+ out = 60 mM). A rate of 250 s−1 was observed with the reduced enzyme (85 s−1 in the absence of Δφ). A rate of 50 s−1 was observed with the oxidized enzyme under the same conditions (15 s−1 in the absence of Δφ). The reconstituted enzyme contained 2 ATPbound per CF0F1 and 1 ADPbound per CF0F1. Upon energisation the enzyme was activated and 0.9 ADP per CF0F1, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 105 M−1·s−1 under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.

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