Gastric H,K-ATPase topography: amino acids 888-907 are cytoplasmic

Biochem Biophys Res Commun. 1991 Nov 14;180(3):1356-64. doi: 10.1016/s0006-291x(05)81345-2.

Abstract

Gastric acidification is mediated by H,K-ATPase, an integral protein of apical membranes of gastric parietal cells. Hydropathy analysis of H,K-ATPase alpha subunit primary structure predicts eight transmembrane (TM) domains, while omeprazole-binding data were interpreted in terms of ten TM domains (Mercier et al. (1991) FASEB J. 5, A749). In the present study, tryptic hydrolysis of gastric mucosal microsomes gave a set of peptides which bound the monoclonal antibody HK 12.18, a highly specific probe of the H,K-ATPase. An antiserum against the C-terminus of H,K-ATPase alpha subunit bound the same peptides, and one smaller peptide. The binding data suggested a putative epitope for HK 12.18, and a 20-mer peptide encompassing this site was synthesized. This peptide bound directly to HK 12.18, displaced HK 12.18 from microsomal H,K-ATPase, and blocked HK 12.18 immunostaining of gastric parietal cells. In addition, intact gastric microsomes competitively inhibited binding of HK 12.18 to peptide-BSA conjugate. Taken together, these data place the HK 12.18 epitope between amino acids 888-907 and identify this domain as cytosolic. This result specifically excludes a pair of TM domains between the sixth and seventh TM alpha helices of the H,K-ATPase and supports a secondary structure model with eight TM domains.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / chemistry*
  • Adenosine Triphosphatases / immunology
  • Amino Acid Sequence
  • Animals
  • Antibodies
  • Cell Membrane / enzymology
  • Enzyme-Linked Immunosorbent Assay
  • Gastric Mucosa / cytology
  • Gastric Mucosa / enzymology
  • H(+)-K(+)-Exchanging ATPase
  • Immunoblotting
  • Immunohistochemistry
  • Molecular Sequence Data
  • Parietal Cells, Gastric / enzymology*
  • Peptide Fragments / isolation & purification
  • Peptides / chemical synthesis
  • Peptides / immunology
  • Swine
  • Trypsin

Substances

  • Antibodies
  • Peptide Fragments
  • Peptides
  • Trypsin
  • Adenosine Triphosphatases
  • H(+)-K(+)-Exchanging ATPase