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

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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 a-catalytic- and a b-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 the b-subunit bears an excess mass of 9 Da attributable to glycosylation. From chemical analysis, there are 0.25 g phospholipids and around 0.024 g cholesterol bound per g protein. Analytical ultracentrifugation shows one main complex, sedimenting at s20w = 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 a,b-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an a,b-protomer with 0.9-1.4 g/g bound detergent and lipids and a reasonable frictional ratio of 1.5 corresponding to Rs= 7.1 nm. An a2,b2 dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H+,K+-ATPase. Thus, a,b 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.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind287
Sider (fra-til)41963-41978
Antal sider15
ISSN0021-9258
DOI
StatusUdgivet - 7 dec. 2012

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