Na+ binding of V-type Na+-ATPase in Enterococcus hirae

Rotation catalysis theory has been successfully applied to the molecular me chanism of the ATP synthase (F0F1-ATPase) and probably of the vacuolar ATPa se. We investigated the ion binding step to Enterococcus hirae Na+-transloc ating V-ATPase, The kinetics of Na+ binding to purified V-ATPase suggested 6 +/- 1 Na+ bound/ enzyme molecule, with a single high affinity (Kd(Na+) = 15 +/- 5 mu M). The number of cation binding sites is consistent with the m odel that V-ATPase proteolipids form a rotor ring consisting of hexamers, e ach having one cation binding site. Release of the bound Na-22(+) from puri fied molecules in a chasing experiment showed two phases: a fast component (about two-thirds of the total amount of bound Na+; k(exchange) > 1.7 min(- 1)) and a slow component (about one-third of the total; k(exchange) = 0.16 min(-1)), which changes to the fast component by adding ATP or ATP gamma S, This suggested that about two-thirds of the Na+ binding sites of the Na+-A TPase are readily accessible from the aqueous phase and that the slow compo nent is important for the transport reaction.