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

Ingrid Dach,Claus Olesen,Luca Signor,Poul Nissen,Marc Le Maire,Jesper V Møller,Christine Ebel

doi:10.1074/jbc.m112.398768

Abstract

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 α-catalytic and a β-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 C(12)E(8), followed by exchange of C(12)E(8) with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-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 α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-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 α(2),β(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H(+),K(+)-ATPase. Thus, α,β 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, Ca(2+)-ATPase and Na(+),K(+)-ATPase.

Highlights

The proton pump Hϩ,Kϩ-ATPase is responsible for the acidification of the gastric fluid
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
With Tween 20. 2.4 sucrose, 3 mM Mg2ϩ, mg of the tubuloveand 1 mM EDTA, was solubilized with 14 mg of C12E8, and non-solubilized residues were removed by centrifugation for 35 min at 100,000 ϫ g at 4 °C. 0.5 ml of the supernatant was loaded onto a 10 ϫ 300-mm Superose 6 column, equilibrated, and eluted at a rate of 0.25 ml/min at 4 °C in the above buffer, not containing C12E8 but with Tween 20 at 2 mg/ml (A) or with C12E8 at 2 mg/ml but without Tween 20 (B)

Summary

Introduction

The proton pump Hϩ,Kϩ-ATPase is responsible for the acidification of the gastric fluid. 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 ␣-catalytic and a ␤-glycosylated subunit. 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. Analytical ultracentrifugation shows one main complex, sedimenting at s20,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 ␣,␤-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an ␣,␤-protomer with 0.9 –1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of

Methods

Results

Conclusion