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

Abstract

Rotation catalysis theory has been successfully applied to the molecular mechanism of the ATP synthase (F0F1-ATPase) and probably of the vacuolar ATPase. We investigated the ion binding step to Enterococcus hirae Na+- translocating V-ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 ± 1 Na+ bound/enzyme molecule, with a single high affinity (K(d(Na+)) = 15 ± 5 μM). The number of cation binding sites is consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. Release of the bound 22Na+ from purified 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 ATPγS. This suggested that about two-thirds of the Na+ binding sites of the Na+- ATPase are readily accessible from the aqueous phase and that the slow component is important for the transport reaction.