ΔpH-activation of the thiol-modified chloroplast ATP hydrolase: Nucleotide binding effects

Abstract

The activation of the membrane-bound ATP hydrolase by the electrochemical proton gradient Δ \\ ̃ gm H + was studied with dithiothreitol-treated, dark-adapted lettuce thylakoids. After reactivation, carried out at different light intensities, hydrolysis of ATP or GTP was measured upon fast membrane deenergization and substrate addition. The following results were obtained. (1) The initial rate of ATP hydrolysis depends on both the ΔpH attained during the light-reactivation stage and on the external pH (ΔpH was measured by the 9-aminoacridine technique, Δψ being collapsed by valinomycin). (2) When hexylamine is present the initial rate of ATP hydrolysis, as that of GTP hydrolysis measured in the presence of 50 μM GDP, only depends on the ΔpH. (3) ADP addition is able to inhibit a fraction of the ATPases within less than a few seconds. The affinity for the inhibitor is increased if ADP is added 10 s after complete membrane deenergization. From the results obtained, with either ATP or GTP in delocalized conditions (hexylamine present), it is proposed that the number of active thiol-reduced ATPases is a simple function of the ΔpH, independent of the external pH. The results without hexylamine are interpreted as being due to an incomplete delocalization of the proton gradient. The mechanistic implications of this ΔpH-activation are discussed. The rapid deactivation of only a fraction of active ATPases by ADP binding, and more especially the decrease in affinity for this inhibitor by ΔpH, suggests the existence of different active states which are discriminated by their deactivation and not their catalytic properties.