Anion-proton cotransport through the human red blood cell band 3 protein. Role of glutamate 681.
The band 3 protein of the human red blood cell membrane contains a glutamate residue that must be protonated in order for divalent (SO4=) anion transport to take place at an appreciable rate. The carboxyl side chain on this glutamate residue can be converted to the primary alcohol by treatment of intact cells with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate) followed by reductive cleavage with BH4-. Edman degradation of CNBr fragments from band 3 labeled in intact cells with Woodward's reagent K and [3H]BH4- showed that Glu681 is heavily labeled under conditions in which Cl- exchange is inhibited, SO4= exchange is accelerated, and Cl- conductance is accelerated. No other glutamate residue in band 3 is detectably labeled under the conditions of these experiments, as demonstrated either by Edman degradation or by the lack of label in major known proteolytic fragments. It is concluded that Glu681 is the binding site for the H+ that is transported with SO4= during band 3-catalyzed H+/SO4= cotransport. This residue is conserved among all species of red cell band 3 (AE1) as well as the related proteins AE2 and AE3. Glu681 is the first amino acid residue in band 3 which has been identified as a binding site for a transported substrate (H+). The functional characteristics of this residue suggest that it lies within the transport pathway and can be alternately exposed to the intracellular and extracellular media.
Jennings
M L
ML
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
Smith
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000838:Anions; D002268:Carboxypeptidases; D002712:Chlorides / Q000097:blood; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011522:Protons; D012689:Sequence Homology, Nucleic Acid; D013431:Sulfates / Q000097:blood
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D002712:Chlorides; D005971:Glutamates; D007555:Isoxazoles; D010446:Peptide Fragments; D011522:Protons; D013431:Sulfates; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D018698:Glutamic Acid; D002268:Carboxypeptidases; C022633:serine carboxypeptidase