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

Although the reversible dissociation of the V(1)V(O) holoenzyme into its V(1) and V(O) 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 V(1)V(O) holoenzyme and of the V(1) complex, both purified from Manduca sexta larval midgut, we found that the V(1) complex contained 1.7 molec. of ADP, whereas only 0.3 molec. of ADP were bound to the V(1)V(O) holoenzyme. By contrast, both proteins contained only negligible amounts of ATP. Incubation of the V(1)V(O) holoenzyme with various adenine nucleotides revealed that ATP hydrolysis, leading to a state containing tightly bound ADP is necessary for its dissociation.