Gastric H,K-ATPase topography: amino acids 888-907 are cytoplasmic.
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.
Smolka
A
A
Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston 29425.
Alverson
L
L
Fritz
R
R
Swiger
K
K
Swiger
R
R
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000166:cytology / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010295:Parietal Cells, Gastric / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D013552:Swine; D014357:Trypsin
D000906:Antibodies; D010446:Peptide Fragments; D010455:Peptides; D014357:Trypsin; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase