CHARACTERIZATION OF THE BETA-SUBUNIT OF THE H+-K+-ATPASE USING AN INHIBITORY MONOCLONAL-ANTIBODY

The gastric proton pump, H+-K+-ATPase, is composed of alpha- and beta-subunits. The 95-kDa alpha-subunit has been referred to as the catalytic subunit containing sites for ATP binding and phosphorylation. The beta-subunit is a glycoprotein with a 34-kDa core peptide that has a single transmembrane segment, a small cytoplasmic NH2-terminal, and a large extracellular COOH-terminal domain with seven potential N-linked glycosylation sites. To further study the beta-subunit, we developed monoclonal antibodies that identified a 52-kDa mannose-rich glycoprotein that was deglycosylated by endoglycosidase H such that six transient intermediates were identified, as well as a 34-kDa beta-subunit core peptide. These observations suggest that the beta-subunit is synthesized as a 52-kDa glycoprotein with seven N-linked precursor high-mannose oligosaccharides that mature into complex oligosaccharides. One antibody, 2G11, inhibits the K+-stimulated ATP hydrolysis as well as K+-stimulated p-nitrophenyl phosphatase (pNPPase) activity of the H+-K+-ATPase. Kinetic studies revealed that 2G11 inhibited maximum velocity (V(max)) of ATP hydrolysis by approximately 50% with no change in the K(m) for K+, whereas, for pNPPase both V(max) and K(m) were altered. These studies demonstrate a functional role for the beta-subunit in the H+-K+-ATPase activity, especially the K+-induced conformational states.