Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+/K(+)-ATPase are located on the cytoplasmic side of the membrane.
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 alpha 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 alpha 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 beta subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.
Antolovic
R
R
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Brüller
H J
HJ
Bunk
S
S
Linder
D
D
Schoner
W
W
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000276:immunology*; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002462:Cell Membrane / Q000201:enzymology; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D006735:Horseradish Peroxidase; D007668:Kidney / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014357:Trypsin
D000596:Amino Acids; D000906:Antibodies; D000939:Epitopes; D003208:Concanavalin A; D006735:Horseradish Peroxidase; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase