pH jump-induced phosphorylation of adenosine diphosphate in thylakoidal membranes

The kinetic properties of pH jump-induced phosphorylation in thylakoidal membranes of spinach chloroplasts were investigated, and the following results were obtained. 1. The pH jump-induced P incorporation proceeded linearly with time for at least 2 sec after the start of the reaction. Phosphate was incorporated mainly into ATP. The amounts of incorporation into ADP during 0.1 and 2.0 sec were 0.02 and 0.1 mole/400 moles chl, respectively. The amounts of P incorporation into ADP and the beta-position of ATP during a 2 sec reaction were less than 5 and 0.1% of the total amount of P incorporation, respectively. Even in the absence of added ADP, ATP was formed by the pH jump, but the amount was very small, i.e., less than 1% of that in the presence of a saturating amount of ADP. Formation of ATP was not enhanced by the addition of 0.1 mM AMP, instead of ADP. 2. The dependence of the rate of ATP formation, v, induced by a pH jump from 3.85 to 8.11 on the concentrations of ADP and Pi was given by v=Vopt/[1+psi1/[ADP]) (1 + psi2/[Pi)], where the values of the constants, Vopt, psi1, and psi2 were 14--20 moles/10-6 g chl/sec, 12.5-15 muM and 11-20 mM, respectively, at 0 degrees. 3. The dependence of v on the concentration of protons was given by v=Va/[1 + psi H-a/[H+,-a])-2], and v =Vb/[1 + ([H+,-b]/psiH-b)-2], in the acidic and basic phases, respectively. The values of the constants psi H-a and psi H-b were 10-5.7 and 10-7.9 M, respectively. 4. ATP formation was initiated by adding one of the substrates, ADP or Pi, at various times after after the pH jump in the presence of the other substrate. The rate decreased logarithmically with increase in the time between the pH jump and the start of the reaction. When phosphorylation was initiated by adding Pi after the pH jump in the presence of ADP, the decay constant of v was about 0.08 sec-1, which was one-third of that observed when the order of addition of ADP and Pi was reversed.