Location of a Cytoplasmic Epitope for Monoclonal Antibody HK 12.18 on H,K-ATPase  Subunit Adam J. Smolka, 1 Kellie A. Larsen, and Charles E. Hammond Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Received June 5, 2000 The enzyme responsible for gastric acidification is a heterodimeric ( and  subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes electroneutral exchange of exoplas- mic K  for cytoplasmic H 3 O  . The molecular mecha- nisms of the catalytic exchange reaction are imper- fectly 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 hy- dropathy plot predictions of HK and HK secondary structure. The monoclonal antibody HK 12.18, which labels gastric mucosal parietal cells by immunocyto- chemistry, and which binds to a single M r 94,000 polypeptide by SDS–PAGE immunoblot of gastric mi- crosomes, has been widely used as a specific marker of parietal cells in clinical and cell biological studies of acid secretion, and as a specific HK probe in bio- chemical studies. However, the uncertain location of the HK 12.18 epitope has limited the antibody’s useful- ness as a topology probe. In this study, HK 12.18 im- mune reactivity with native H,K-ATPase tryptic pep- tides, HK cDNA fragments expressed in bacteria, and overlapping synthetic HK tridecapeptides, was used to identify the HK 12.18 epitope as seven consecutive amino acids (Asp 682 -Met-Asp-Pro-Ser-Glu-Leu 688 ) in the cytoplasmic middle third of HK. © 2000 Academic Press Gastric acid secretion is mediated by a Mg 2+ - dependent, K + -stimulated, H + -transporting adenosine triphosphatase (H,K-ATPase, EC 3.6.1.36) (1, 2). The enzyme is located in tubulovesicular and secretory can- alicular membranes in gastric epithelial parietal cells, and catalyzes the electroneutral exchange of lumenal K + for cytoplasmic H + . The resulting million-fold H + gradient across the secretory membranes makes the H,K-ATPase an attractive model for structure-function studies of epithelial proton pumping. The enzyme is a vanadate-sensitive P-type ATPase with closely- interacting  and  subunits. H,K-ATPase  subunits (HK) consist of 1033 amino acids, with a relative molecular mass (M r ) of 94,000 estimated by SDS– PAGE. H,K-ATPase  subunits (HK) consist of 290 – 294 amino acids, and glycosylation at 6 or 7 tripeptide consensus sites gives a diffuse M r 60,000 – 80,000 (3, 4). Antibodies directed against HK and HK have been important tools in confirming many specific pre- dictions of H,K-ATPase secondary structure based on primary structure hydropathy analysis. For HK, sev- eral lines of evidence based on antibody-binding and other data are consistent with cytoplasmic N- and C-termini (5, 6), four transmembrane (Tm)  helices in the N-terminal third of the subunit, a hydrophilic cy- toplasmic domain containing the phosphorylation and nucleotide binding sites, and six C-terminal Tm do- mains (7–9). For HK, immunochemical data confirms exoplasmic orientation of the C-terminus (9), and a single transmembrane domain, located 50 residues from the cytoplasmic N-terminus. These topological data were acquired using a wide range of antibodies, including polyclonal rabbit sera raised against syn- thetic peptides based on HK and HK primary struc- tures (5), monoclonal antibodies against whole parietal cells (10), gastric microsomal membranes (11), or gel- purified single-band preparations of HK (4, 12) and “flu tag” antibodies directed against exogenous hemag- glutinin antigen peptides inserted into HK or HK cDNAs, and subsequently expressed in heterologous cell systems (9). The aim of this study was to define the minimal epitope recognized by the monoclonal antibody HK 12.18, widely used as an immunocytochemical marker for parietal cells, both in situ and in purified cell prep- arations, and as a biochemical probe of HK biosyn- thesis, turnover, regulation, interaction with HK, and Amino acid single-letter abbreviations are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. 1 To whom correspondence should be addressed at Division of Gastroenterology and Hepatology, Department of Medicine, CSB 916, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. Fax: (843) 792 1153. E-mail: smolkaaj@ musc.edu. Biochemical and Biophysical Research Communications 273, 942–947 (2000) doi:10.1006/bbrc.2000.3031, available online at http://www.idealibrary.com on 942 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.