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

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A. N. Malyan

Institute of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; fax: (7-0967) 330-532; E-mail: Malyan@issp.serpukhov.su

Received October 21, 2004; Revision received November 22, 2004

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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 µM. 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.

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KEY WORDS: ATP synthase, CF₀F₁, noncatalytic sites, chloroplasts

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