Epitope mapping by amino‐acid‐sequence‐specific antibodies reveals that both ends of the α subunit of Na+/K+‐ATPase are located on the cytoplasmic side of the membrane

Abstract

Right‐side‐out vesicles of pig kidney microsomes and amino‐acid‐sequence‐specific antibodies were used to probe the sidedness of the C‐terminus and the N‐terminus of the catalytic α subunit of Na+/K+‐ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N‐terminal sequence GRDKYEPAAVSE (peptide 1–12) and against peptides corresponding to the C‐terminal sequences IFVYDEVRKLIIRRR (peptide 991–1005) and RPGGWVEKETYY (peptide 1005–1016). These antibodies were purified by affinity chromatography on the respective peptide‐Sepharose columns. Moreover, antibodies against the N‐terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the α subunit of pork kidney Na+/K+‐ATPase. These antibodies reacted with native as well as SDS‐denaturated Na+/K+‐ATPase. When the antibodies were used to probe the sidedness of the sequences in right‐side‐out vesicles of pig kidney microsomes, the N‐terminal peptide 1–12 as well as the C‐terminal peptides 991–1005 and 1005–1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the β subunit, a glycoprotein, reacted with the outside of right‐side‐out vesicles.