C-TERMINAL TOPOLOGY OF GASTRIC H+,K+-ATPASE

An antibody was prepared against a peptide corresponding to residues 1024-1034 (the putative C-terminus) of the alpha-subunit of hog gastric H+,K+-ATPase. The antibody bound to a 95 kDa band of H+,K+-ATPase that was solubilized in SDS, but not to that of Na+,K+-ATPase. It also bound to products of tryptic digestion that included C-terminal fragments of the H+,K+-ATPase alpha-subunit. The same amount of the antibody bound to both intact (tight) and lyophilized (leaky) inside-out gastric vesicles, indicating that its epitope is present on the cytosolic side of the vesicles. This finding was further confirmed by using fluorescence-immunolocalization techniques and streptolysin-O to permeabilize newt oxyntic cells. Stimulation of isolated newt oxyntic cells with dibutyryl cyclic AMP induces fusion of tubulovesicles with the apical membrane, so that the luminal domains of the H+,K+-ATPase alpha-subunit directly face the cell-suspension medium. The antibody did not bind to the stimulated intact cell, but bound to cells permeabilized with streptolysin-O, indicating that it binds from the cytoplasmic side to the C-terminus of the H+,K+-ATPase alpha-subunit in apical and tubulovesicular membrane, and also that the H+,K+-ATPase alpha-subunit has an even number of transmembrane domains.