ADP BINDING AND ATP SYNTHESIS BY RECONSTITUTED H+-ATPASE FROM CHLOROPLASTS

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 Delta pH (pH(in) = 5.0, pH(out) = 8.5) and a K+/valinomycin diffusion potential, Delta phi (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 Delta phi). A rate of 50 s(-1) was observed with the oxidized enzyme under the same conditions (15 s(-1) in the absence of Delta phi). The reconstituted enzyme contained 2 ATP(bound) per CF0F1 and 1 ADP(bound) 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 10(5) 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.