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

Kazuhiro Abe; Claus Olesen

doi:10.1007/978-1-4939-3179-8_4

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

Methods Mol Biol. 2016:1377:19-27. doi: 10.1007/978-1-4939-3179-8_4.

Authors

Kazuhiro Abe^{1

2}, Claus Olesen^{3

4}

Affiliations

¹ Cellular and Structural Physiology Institute, Nagoya University, Nagoya, 464-8601, Japan. kabe@cespi.nagoya-u.ac.jp.
² Department of Medicinal Science, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, 464-8601, Japan. kabe@cespi.nagoya-u.ac.jp.
³ Department of Molecular Biology and Genetics, Aarhus University, Gustav Wiedsvej 10, 8000, Aarhus C, Denmark.
⁴ Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Gustav Wiedsvej 10, 8000, Aarhus C, Denmark.

PMID: 26695019
DOI: 10.1007/978-1-4939-3179-8_4

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.

Keywords: Gastric proton pump; H+,K+-ATPase; Membrane protein; Purification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Membrane / enzymology*
Centrifugation, Density Gradient
H(+)-K(+)-Exchanging ATPase / chemistry
H(+)-K(+)-Exchanging ATPase / isolation & purification*
Molecular Biology / methods*
Stomach / enzymology*
Swine

Substances

H(+)-K(+)-Exchanging ATPase