ACTIVATION AND DEACTIVATION OF THE THIOL-MODULATED CHLOROPLAST H+-ATPASE DURING ATP HYDROLYSIS

Formation of a transmembrane proton gradient, tight binding of adenine nucleotides and ATPase activity were followed during hydrolysis of a limited amount of ATP by thylakoids preilluminated in the presence of DTT. The following results were obtained: (1) While a micromolar concentration of ADP caused complete deactivation of the ATPase within 15 s, the same concentration of ATP sustained enzyme activity for more than 1 min due to a small proton gradient generated by hydrolysis of the ATP. (2) In the presence of ADP, steady-state ATPase activity and levels of tightly bound nucleotides are inversely influenced by ΔpH. When ADP is replaced by ATP, the activity curve is shifted towards much lower ΔpH values and is not significantly different from the ΔpH curve obtained in the absence of medium nucleotides. (3) At low medium ATP concentration (5 μM), ATP as well as ADP is incorporated into membrane-bound CF1; the ratio of tightly bound ATP ADP increases while the sum of ATP plus ADP decreases with increasing ΔpH. An enzyme with a tightly bound ATP can be active or inactive, depending on the actual ΔpH. (4) Hydrolysis of ATP by thylakoids containing a certain percentage of active ATPases, effects extensive reactivation of inactive enzyme molecules due to coupled ΔpH formation. At exhaustion of the substrate, however, the total enzyme population is inactivated within 1 min. Inactivation is a synergic result of the decay of the proton gradient and tight binding of the hydrolysis product ADP. © 1990.