Relation between the initial kinetics of ATP synthesis and of conformational changes in the chloroplast ATPase studied by external field pulses

Abstract

ATP formation and the energy-dependent release of tightly bound [ 14C]-adenine nucleotides from the chloroplast coupling factor CF 1 has been studied as a function of the time of energization of the membrane in the range of 500 μs up to 60 ms. The high time resolution was achieved because the energization was generated artificially by external electric field pulses. Applying external electric field pulses to a chloroplast suspension induces an electric potential difference across the thylakoid membrane. The following results were obtained: 1. (1) The amount of ATP generated increases linearly with the time of energization. The steady-state rate of ATP formation is reached in less than 500 μs. 2. (2) A fraction of the adenine nucleotides tightly bound to CF 1 is released on energization with a half-rise-time of about 2 ms. The size of the fraction, i.e., the amplitude of the fast phase of the release, increases with the magnitude of the induced transmembrane electric potential difference. A further slow release is superimposed. 3. (3) The initial rate of the release of adenine nucleotides is practically identical with the rate of ATP formation. It is concluded that the release of tightly bound nucleotides monitors an initial conformational change by which the ATPase turns from an inactive into an activated state. For the explanation of the results a reaction scheme is proposed which takes into account a preceding activation of the ATPase.