Use of HPLC for the study of ADP binding to chloroplast ATPase. II. Its effect on enzymatic activity

The binding of ADP to chloroplast ATPase (CF1) has been measured by the chromatographic method of Hummel and Dreyer which allows low affinity binding determinations. Besides about 1.5 endogenous ADP molecules that are irreversibly bound to CF1 or slowly exchangeable in the experimental conditions, ADP binds to CF1 in the presence of Mg2+ with an apparent unique dissociation constant of 64 mu M, up to a total of 6 +/- 0.5 moles/mole, when Mg2+ is in excess over ADP. Under these conditions, the major part of the bound nucleotide is in the metal complexed form. Metal free ADP also binds to CF1, with a dissociation constant of 5 to 15 mu M, measured by competitive inhibition of ATPase activity, at low Mg2+ concentration. ADP and ATP appear to bind competitively on CF1 the extent of binding of each nucleotide can be shown and measured by the chromatographic method of Hummel and Dreyer, using an anion exchange column which separates ADP from ATP. The fractions of each nucleotide have been calculated, using the dissociation constants determined in the conditions of the measurement.