Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythrocyte membrane.
Band 3 is the major, membrane-spanning, approximately90 000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, we have cleaved this protein in situ into large fragments and ascertained their disposition. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38 000 and 55 000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they are anchored in the apolar core of the membrane. Both pieces communicate with the extracellular space, and the 55 000 dalton species extends to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17 000 dalton species, a segment of the 55 000 dalton fragment, which spans and is firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52 000 daltons and released major band 3 fragments of less than or equal to 41 000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which can be induced only at the cytoplasmic surface, cross-linked intact band 3, and certain of its water-soluble fragments. Finally, fragments of 23 000 daltons were generated from the innersurface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.
D000595:Amino Acid Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism*; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000097:blood*; D011485:Protein Binding; D011487:Protein Conformation
D004220:Disulfides; D010446:Peptide Fragments; D010455:Peptides; D010206:Papain