Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes. Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells. Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit. It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain. The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region. The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions. This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions. The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments.
Amino Acid Sequence Animals