Isolation of H(+),K(+)-ATPase-enriched Membrane Fraction from Pig Stomachs

Gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for the acid secretion. Here, we describe the procedure for the isolation of H(+),K(+)-ATPase-enriched membrane vesicle fractions by Ficoll/sucrose density gradient centrifugation. Further purification by SDS treatment of membrane fractions is also introduced. These procedures allow us to obtain purified protein preparations in a quantity of several tens of milligrams, with the specific activity of ~480 μmol/mg/h. High purity and stability of H(+),K(+)-ATPase in the membrane preparation enable us to evaluate its detailed biochemical properties, and also to obtain 2D crystals for structural analysis.