Location of a Cytoplasmic Epitope for Monoclonal Antibody HK 12.18 on H,K-ATPase α Subunit

Abstract

The enzyme responsible for gastric acidification is a heterodimeric (α and β subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes electroneutral exchange of exoplasmic K+ for cytoplasmic H3O+. The molecular mechanisms of the catalytic exchange reaction are imperfectly understood, and await clarification of the precise topology of the enzyme with respect to the secretory membrane. Antibodies directed against H,K-ATPase subunits have been useful in confirming hydropathy plot predictions of HKα and HKβ secondary structure. The monoclonal antibody HK 12.18, which labels gastric mucosal parietal cells by immunocytochemistry, and which binds to a single Mr ∼94,000 polypeptide by SDS–PAGE immunoblot of gastric microsomes, has been widely used as a specific marker of parietal cells in clinical and cell biological studies of acid secretion, and as a specific HKα probe in biochemical studies. However, the uncertain location of the HK 12.18 epitope has limited the antibody's usefulness as a topology probe. In this study, HK 12.18 immune reactivity with native H,K-ATPase tryptic peptides, HKα cDNA fragments expressed in bacteria, and overlapping synthetic HKα tridecapeptides, was used to identify the HK 12.18 epitope as seven consecutive amino acids (Asp682-Met-Asp-Pro-Ser-Glu-Leu688) in the cytoplasmic middle third of HKα.