title: Isolation of H(+),K(+)-ATPase-enriched Membrane Fraction from Pig Stomachs.
abstract: 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.
authors.affiliation: Cellular and Structural Physiology Institute, Nagoya University, Nagoya, 464-8601, Japan. kabe@cespi.nagoya-u.ac.jp.|Department of Medicinal Science, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, 464-8601, Japan. kabe@cespi.nagoya-u.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Abe
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Gustav Wiedsvej 10, 8000, Aarhus C, Denmark.|Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Gustav Wiedsvej 10, 8000, Aarhus C, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Olesen
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000302:isolation & purification*; D008967:Molecular Biology / Q000379:methods*; D013270:Stomach / Q000201:enzymology*; D013552:Swine
chemical_list: D017506:H(+)-K(+)-Exchanging ATPase
keywords: Gastric proton pump; H+,K+-ATPase; Membrane protein; Purification