Localization of the carboxyl terminus of Band 3 to the cytoplasmic side of the erythrocyte membrane using antibodies raised against a synthetic peptide.
Polyclonal antibodies were raised in rabbits against a synthetic peptide which corresponds to the 12-amino acid carboxyl-terminal sequence of murine erythrocyte Band 3. Immunoblots of ghost membrane proteins showed that the antibody specifically recognized murine or rat Band 3 but not human or canine Band 3. The antibody also bound to murine ghost membranes applied directly to nitrocellulose but not to human ghost membranes. This shows that the carboxyl terminus of Band 3 is available for antibody binding in ghost membranes and that the carboxyl-terminal sequences of human and mouse Band 3 are not identical. The specificity of the antibody for the carboxyl terminus of Band 3 was confirmed by the loss of antibody binding after digestion of detergent-solubilized ghost membrane proteins with carboxypeptidase Y. In addition, carboxyl-terminal fragments of Band 3 generated by protease treatment of cells or ghost membranes were positive on immunoblots while amino-terminal fragments were negative. In contrast, protease-treated stripped ghost membranes did not contain a carboxyl-terminal fragment of Band 3 that was detectable on immunoblots. The carboxyl terminus of Band 3 was localized to the cytoplasmic side of the erythrocyte membrane since antibody binding as determined by immunofluorescence occurred in ghosts and permeabilized cells but not in intact cells. In addition, competition studies using enzyme-linked immunosorbent assays and immunoblots showed that cells and resealed ghosts competed poorly for antibody compared to ghost membranes, inside-out vesicles, or albumin-conjugated peptide.
Lieberman
D M
DM
Department of Medicine, University of Toronto, Ontario, Canada.
Reithmeier
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001667:Binding, Competitive; D002268:Carboxypeptidases / Q000378:metabolism; D003593:Cytoplasm; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007118:Immunoassay; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D013045:Species Specificity
D001457:Anion Exchange Protein 1, Erythrocyte; D000906:Antibodies; D000941:Antigens; D010446:Peptide Fragments; D002268:Carboxypeptidases; C022633:serine carboxypeptidase