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Active detergent-solubilized H+,K+-ATPase is a monomer.

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 ?-catalytic and a ?-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 ?-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 ?,?-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 1.5, corresponding to a Stokes radius of 7.1 nm. An ?(2),?(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H(+),K(+)-ATPase. Thus, ?,? 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.

SUBMITTER: Dach I

PROVIDER: S-EPMC3516743 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Active detergent-solubilized H+,K+-ATPase is a monomer.

Dach Ingrid I Olesen Claus C Signor Luca L Nissen Poul P le Maire Marc M Møller Jesper V JV Ebel Christine C

The Journal of biological chemistry 20121010 50

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), follow ...[more]

PMID: 23055529

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Project description:Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

Dataset Information

Active detergent-solubilized H+,K+-ATPase is a monomer.

Publications

Active detergent-solubilized H+,K+-ATPase is a monomer.

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