ANTIBODY EPITOPE MAPPING OF THE GASTRIC H+ K+-ATPASE/

Several antibodies against the gastric H+/K+-ATPase were analysed for the topological and sequence location of their epitopes. Topological m apping was done by comparing indirect immunofluorescent staining in in tact and permeabilised rat parietal cells. Epitope definition was by W estern analysis of intact and of trypsin or V8-proteinase-fragmented h og gastric ATPase combined with N-terminal sequencing of the fragments ; by Western analysis of fragments of rabbit alpha subunit expressed i n Escherichia coli; by analysis of rabbit alpha and beta subunits expr essed in baculovirus-transfected SF 9 cells and by ELISA assay of synt hetic octamers of one region of the hog alpha subunit. It was confirme d that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic r egion between M4 and M5, close to the ATP-binding domain. The major ep itope 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 m onoclonal antibody, mAb 146-14, was shown to recognise an extracytopla smic epitope dependent on intact disulfide bonds, present in the rat a nd the rabbit, but absent in the hog beta subunit, due to non-conserva tive amino-acid substitutions. This antibody also recognised an epitop e present in the alpha subunit of the H+/K+-ATPase at the M7 extracyto plasmic interface, perhaps indicating structural association of these two regions. The polyclonal antibody, pAb39, raised against the C-term inal 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.