Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [14C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF1. [14C]ADP binding exhibits a monophasic concentration curve with a Km of 2 micronM. 2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF1 conformation is established. 3. Re-binding of [14C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF1, [14C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF1 is converted to a non-exchangeable form. 4. Energy-dependent adenine nucleotide exchange on CF1 is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.