Active Detergent-solubilized H+,K+-ATPase Is a Monomer

The H+,K+-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an alpha-catalytic and a beta-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H+,K+-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C12E8, followed by exchange of C12E8 with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the beta-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s(20,w) = 7.2 +/- 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H+,K+-ATPase alpha,beta-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an alpha,beta-protomer with 0.9-1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An alpha(2),beta(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H+,K+-ATPase. Thus, alpha,beta H+,K+-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca2+-ATPase and Na+,K+-ATPase.