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 (Vmax) of ATP hydrolysis by approximately 50% with no change in the Km for K+, whereas, for pNPPase both Vmax and Km were altered. These studies demonstrate a functional role for the beta-subunit in the H(+)-K(+)-ATPase activity, especially the K(+)-induced conformational states.
Chow
D C
DC
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Forte
J G
JG
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015153:Blotting, Western; D003488:Cyanogen Bromide; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000166:cytology / Q000201:enzymology*; D006031:Glycosylation; D017506:H(+)-K(+)-Exchanging ATPase / Q000032:analysis*; D007150:Immunohistochemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000037:antagonists & inhibitors; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011188:Potassium / Q000494:pharmacology; D054328:Proton Pump Inhibitors; D011817:Rabbits; D013552:Swine
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D054328:Proton Pump Inhibitors; D009597:4-Nitrophenylphosphatase; D017506:H(+)-K(+)-Exchanging ATPase; D003488:Cyanogen Bromide; D011188:Potassium