Modulation of coupling factor ATPase activity in intact chloroplasts. Reversal of thiol modulation in the dark

Abstract

Illumination of intact pea chloroplasts results in both the pH activation and the thiol modulation of the reversible protonmotive ATPase, or CF 0-CF 1 (Mills, J.D., Mitchell, P. and Schürmann, P. (1980) FEBS Lett. 112, 173–177). We have studied the reversal of the activation and thiol-modulation processes that occurs when illuminated chloroplasts are darkened. (1) Methyl viologen is shown to prevent light-dependent thiol modulation of CF 0-CF 1 in intact chloroplasts. Studies using methyl viologen indicate that the decline in ATPase activity of intact chloroplasts in the dark is due both to pH deactivation and thiol demodulation of CF 0-CF 1. Reillumination in the presence of methyl viologen reactivates CF 0-CF 1 complexes that have undergone pH deactivation, but does not allow thiol modulation to occur. (2) At 20°C, both pH deactivation and thiol demodulation of CF 0-CF 1 are complete within 10 min darkness. At 3.5°C, both processes are retarded, and require more than 30 min for completion. (3) Lysis of intact chloroplasts greatly retards the thiol demodulation of CF 0-CF 1. In the dark, addition of oxidants to lysed chloroplasts promotes thiol demodulation. Under these conditions, thioredoxin behaves as a weak oxidant, but glutathione has very little effect. Illumination of intact chloroplasts followed by rapid lysis and storage on ice stabilises the pH-activated, thiol-modulated state of CF 0-CF 1 for extended periods in the dark and increases the time required for thiol demodulation to well over 2 h. This method may prove to be useful for preparing thiol-modulated chloroplasts without the need to add or remove exogenous thiols. The results suggest that an oxidative system exists in the stroma which actively reverses thiol modulation of CF 0-CF 1 in the dark. Such a system would be expected if thiol modulation of CF 0-CF 1 were an important physiological process regulating reversible ATPase activity in vivo.