Phosphatase activity of H+-ATPase from chloroplasts

AbstractThe chloroplast H+-ATPase (CF0F1) was purified from spinach chloroplasts and studied both in the soluble state and after reconstitution into asolectin liposomes. Hydrolysis of ATP and of p-nitrophenylphosphate (p-NPP) catalysed by CF0F1 was investigated. (1) Soluble, isolated CF0F1 catalyzes ATP hydrolysis and p-NPP hydrolysis. (2) ATP inhibits the phosphatase activity in the latent state (Ki=1.7 mM). (3) Addition of 100 mM sulfite increases the rate of ATP hydrolysis by a factor of 10, while p-NPP hydrolysis is completely abolished. (4) CF0F1 reconstituted into asolectin vesicles catalyzes ATP hydrolysis and p-NPP hydrolysis. When the enzyme is brought into its active state by a ΔpH/Δϕ jump, ATP hydrolysis is increased by a factor of 8, and p-NPP hydrolysis is completely abolished. (5) ATP hydrolysis by the activated enzyme is inhibited by p-NPP (Ki=1.6 mM). (6) p-NPP also inhibits ATP synthesis by the activated enzyme, competing with phosphate (Ki=0.9 mM). These results show that in the active state of CF0F1, p-NPP is not hydrolyzed but acts as a competitive inhibitor; in the inactive state of CF0F1, p-NPP is hydrolyzed. Hydrolysis of p-NPP might be used as an assay for the inactive forms of CF0F1