ΔpH-dependent activation of chloroplast coupling factors and external pH effects on the 9-aminoacridine response in lettuce and spinach thylakoids

Abstract

Investigation of steady-state ΔpH and photophosphorylation in envelope-free chloroplasts extracted from lettuce or spinach leaves gave the following results. (1) The ratio of the rates of photophosphorylation at two nucleotide concentrations but at the same ΔpH adjusted by light intensity, and measured by 9-aminoacridine distribution, is independent of the magnitude of this ΔpH. Thus the flow-force relationships only represent the Δ μ H + activation of the coupling factors, each activated enzyme running at its maximum turnover rate. (2) Flow-force curves at pH 8.0 and pH 8.5 have similar shapes when ΔpH is delocalized with 200 μM hexylamine. Thus, activation of the coupling factors (CF) depends on the absolute value of ΔpH, regardless of the respective external and internal pH. This would mean that the basic process of activation is either voltage-dependent, or is due to the strictly coupled deprotonation of an external site of CF and protonation of an internal group. (3) The above result is not modified when GDP is used instead of ADP. This suggests that the shift in the equilibrium between active and inactive CF, due to tight binding of ADP, does not depend on the external pH. (4) With lettuce thylakoids, flow-force curves at pH 8.0 and 8.5 become identical only after a translation of the former by +0.15 along the ΔpH axis. Thus the response of 9-aminoacridine depends on the external pH, probably due to a change in the osmotic volume. The internal volume would be 40% higher at pH 8.5 than at pH 8.0. (5) Shifting the external pH from 8.0 to 8.5 has the same effect on 9-aminoacridine and imidazole responses. This confirms that the main effect is an increase of the lumenal volume at high pH. Direct estimations of this volume are qualitatively consistent with this view. (6) With spinach thylakoids, the effect of external pH on the 9-aminoacridine response is minimized. The 9-aminoacridine space change would be only 17% between pH 8.5 and pH 8.0. At pH 8.0, the light-induced uptake of 9-aminoacridine by lettuce thylakoids is insensitive to the increase in sorbitol concentration above 200 mM, whereas it decreases, as expected, with spinach thylakoids. (7) When the ΔpH is delocalized with 200 μM hexylamine, ΔpH and phosphorylation rates at low light intensities do not depend on the external pH. This means that not only the coupling factors, but also basal leaks, would be regulated by ΔpH and not by internal pH or a complex combination of external and internal pH. The results are discussed with reference to previous reports on the pH and ΔpH dependency of ATP synthesis by thylakoids. It seems likely that most early discrepancies were due to the different origins of the chloroplasts and to specific behaviour with respect to the ΔpH probe. This should be considered as a warning when one compares data obtained with membranes from different plant species.