Influence of ATP and ADP on dissociation of the V-ATPase into its V1 and VO complexes

Although the reversible dissociation of the V1VO holoenzyme into its V1 and VO complexes is a general mechanism for the regulation of V-ATPases, important aspects are still not understood. By analyzing the endogenous nucleotide content of the V1VO holoenzyme and of the V1 complex, both purified from Manduca sexta larval midgut, we found that the V1 complex contained 1.7 molec. of ADP, whereas only 0.3 molec. of ADP were bound to the V1VO holoenzyme. By contrast, both proteins contained only negligible amounts of ATP. Incubation of the V1VO holoenzyme with various adenine nucleotides revealed that ATP hydrolysis, leading to a state containing tightly bound ADP is necessary for its dissociation.