Site-directed antibodies as topographical probes of the gastric H,K-ATPase alpha-subunit.
Gastric acid is secreted by an ATP-driven H+ and K+ exchanger (H,K-ATPase), an integral apical membrane protein of parietal cells. Although the primary structure of the enzyme is known, its higher order structure is uncertain. In order to acquire topographical probes of native, microsomal H,K-ATPase, synthetic peptides corresponding to the 17 amino-terminal (N-peptide) and 16 carboxyl-terminal (C-peptide) residues of pig gastric H,K-ATPase alpha-subunit were coupled to keyhole limpet hemocyanin (KLH). Rabbits were immunized with peptide-KLH conjugates and their sera were tested for specificity by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunocytochemistry. All sera showed high ELISA reactivities with synthetic peptides, peptide-BSA conjugates, and microsomal H,K-ATPase adsorbed to microtiter wells (some titers greater than 1:10(4)). Immunoblots of H,K-ATPase resolved by SDS-PAGE showed both N-peptide and C-peptide antibodies reacting with a single 94 kDa band. All sera selectively stained parietal cells in pig gastric mucosal sections. Preimmune sera gave negative or weak signals in all assays. In competition ELISAs, N-peptide antibodies, but not C-peptide antibodies, were displaced from the corresponding bound synthetic peptides by added microsomal H,K-ATPase. One of the N-peptide antibodies inhibited H,K-ATPase activity by more than 50%; binding of this antibody was decreased when ATP or K+ were bound to the enzyme. These results indicate a cytoplasmically-oriented alpha-subunit N-terminus which may participate conformationally in the H,K-ATPase catalytic cycle, and suggest that antibodies against synthetic H,K-ATPase peptides are potentially useful probes of native microsomal H,K-ATPase topography.
Smolka
A
A
Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston 29425.
Swiger
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000201:enzymology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D027961:Potassium-Hydrogen Antiporters; D013552:Swine
D000906:Antibodies; D002352:Carrier Proteins; D027961:Potassium-Hydrogen Antiporters