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 α-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 α-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 α-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 α-subunit in apical and tubulovesicular membrane, and also that the H⁺,K⁺-ATPase α-subunit has an even number of transmembrane domains.