title: Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes.
abstract: The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D014633:Valine / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001457:Anion Exchange Protein 1, Erythrocyte; D010446:Peptide Fragments; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D014633:Valine