Monoclonal antibody HK4001 completely inhibits K+-dependent ATP hydrolysis and H+ Transport of hog gastric H+K+-ATPase

A monoclonal antibody (designated as HK4001) was prepared against hog gastric H⁺, K⁺-ATPase. It dose-dependency inhibited the H⁺, K⁺-ATPase activity, formation of the K⁺-sensitive phosphoenzyme, and proton uptake into gastric vesicles. The H⁺, K⁺-ATPase activity was completely inhibited by addition of the antibody at a molar ratio of 1: 2 (antibody/catalytic subunit) at pH 7.8. The maximal inhibition decreased with decrease in pH of the medium (7.8 > 7.4 > 6.2). The Fab fragment obtained by digestion of the antibody with papain was also inhibitory. The antibody did not inhibit the K'-dependent p-nitrophenylphosphatase or the labeling of the enzyme with fluorescein isothiocyanate. It inhibited gastric H⁺, K⁺-ATPase from rabbits and rats, but did not cross-react with related cation-transport ATPases (Na⁺, K⁺-ATPase or Ca²⁺-ATPase) or H⁺-ATPase in the multi-vesicular body. From these and related findings, the antibody was suggested to recognize a highly specific site on the cytosolic surface of H⁺, K⁺-ATPase. The conformation of the epitope was conserved after treatment with Triton X-100, but not sodium dodecyl sulfate. In addition, judging from the stoichiometry of inactivation of H⁺, K⁺-ATPase by this antibody, the functional unit of H⁺, K⁺-ATPase was suggested to be a dimer or a tetramer (not a trimer) of the catalytic unit.