Light-Dependent Incorporation of Adenine Nucleotide into Noncatalytic Sites of Chloroplast ATP Synthase

The binding of ADP and ATP to noncatalytic sites of dithiothreitol-modified chloroplast ATP synthase was studied. Selective binding of nucleotides to noncatalytic sites was provided by preliminary light incubation of thylakoid membranes with 14C]ADP followed by its dissociation from catalytic sites during dark ATP hydrolysis stimulated by bisulfite ions ("cold chase"). Incorporation of labeled nucleotides increased with increasing light intensity. Concentration-dependent equilibrium between free and bound nucleotides was achieved within 2-10 min with the following characteristic parameters: the maximal value of nucleotide incorporation was 1.5 nmol/mg of chlorophyll, and the dissociation constant was 1.5 microM. The dependence of nucleotide incorporation on Mg2+ concentration was slight and changed insignificantly upon substituting Ca2+ for Mg2+. Dissociation of nucleotide from noncatalytic sites was illumination-dependent. The dissociation kinetics suggested the existence of at least two nucleotide-binding sites with different dissociation rate constants.