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 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.
Center for Membrane Pumps in Cells and Diseases, Danish Research Foundation, DK-8000 Aarhus, Denmark.
Ingrid
I
Dach
Claus
C
Olesen
Luca
L
Signor
Poul
P
Nissen
Marc
M
le Maire
Jesper V
JV
Møller
Christine
C
Ebel
D000818:Animals; D003902:Detergents / Q000737:chemistry*; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification; D020836:Protein Structure, Quaternary; D012995:Solubility; D013552:Swine; D014461:Ultracentrifugation
D003902:Detergents; D017506:H(+)-K(+)-Exchanging ATPase