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-dependently 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 greater than 7.4 greater than 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 multivesicular 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.