Correlation between the activity of membrane-bound ATPase and the decay rate of flash-induced 515-nm absorbance change in chloroplasts in intact leaves, assayed by means of rapid isolation of chloroplasts

Abstract

(1) The relationship between activation of the membrane-bound ATPase and the stimulation of dissipation of the flash-induced membrane potential by preillumination was studied in intact spinach leaves by measuring the ATPase activity of rapidly isolated chloroplasts and the decay of the flash-induced 515-nm absorbance change ( ΔA 515) in intact leaves. (2) The decay of ΔA 515 was accelerated by preillumination. The ΔA 515 decay in leaves treated with N, N′-dicyclohexylcarbodiimide (DCCD) became slower and was not accelerated by preillumination. However, treatment with DCCD did not lower the intensity of delayed fluorescence. (3) Membrane-bound ATPase of chloroplasts which were rapidly isolated from the preilluminated leaves (90 s preparation time) showed a higher activity (over 200 μmol P i/mg chlorophyll per h in the case of 2-min preillumination) than that of chloroplasts isolated from dark-adapted leaves. (4) The acceleration of ΔA 515 decay and the activation of ATPase showed similar dependences on illumination time in intact leaves. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea, carbonyl cyanide p-chlorophenylhydrazone and DCCD inhibited the activation of ATPase and the acceleration of the ΔA 515 decay by preillumination. (5) The ATPase activity of chloroplasts isolated from illuminated leaves showed a single exponential decay (‘dark inactivation in vitro’). The ATPase activity induced by illuminating the leaves became lower as the dark interval between illumination and the isolation of chloroplasts was increased (‘dark inactivation in vivo’). The time course of the decay of activity had a lag and showed a sigmoidal curve when plotted semilogarithmically. The decay had an apparent half-time of 25 min. (6) The recovery of the accelerated ΔA 515 decay in preilluminated leaves to the original slow rate showed a sigmoidal decay similar to that of the activity of ATPase in intact leaves with a half-time of about 23 min in the dark. (7) It was concluded that the decay rate of ΔA 515 reflected the chloroplast ATPase activity in intact leaves and that the ion conductance of thylakoid membrane was mainly determined by the H + flux through the ATPase, the activity of which was increased after the formation of the high-energy state.