Peptides of human erythrocyte band 3 protein produced by extracellular papain cleavage.
The structure of the human red blood cell membrane band 3 protein has been investigated by proteolysis of intact cells with papain. Papain cleaves the 35,000-dalton chymotryptic peptide (CH35) of band 3 into two integral fragments of Mr 7,500 (P7) and 28,000 (P28). The papain cleavage of CH35 causes inhibition of band 3-catalyzed Cl transport. The peptide P28 carries the band 3 carbohydrate and also contains the 2 cysteine residues in CH35. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) reacts covalently with a lysine residue on P28, rather than P7, in native band 3. However, if the cells are first digested with papain and then reacted with H2DIDS, there is significant covalent reaction with P7, producing a covalent cross-link between P7 and the 60,000-dalton chymotryptic peptide (CH60). Graded papain digestion experiments and end group determinations indicate that the alignment of the band 3 peptides is N-CH60-P7-P28-COOH. The NH2-terminal sequence of P7 is identical with a segment of a peptic fragment of band 3 recently sequenced (Brock, C.J., Tanner, M.J.A., and Kempf, C. (1983) Biochem. J. 213, 577-586). This published sequence, plus our sequence data on CH35 and P7, show that papain cleaves the outer surface of CH35 at two sites, which are 6 residues and 71 residues from the chymotrypsin cleavage site. The 65-residue peptide (P7) between these sites is the best characterized segment of band 3 thus far described: its sequence and location in the band 3 primary structure are now known, and both ends of the peptide are unambiguously exofacial.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D000596:Amino Acids / Q000032:analysis; D001457:Anion Exchange Protein 1, Erythrocyte; D004910:Erythrocyte Membrane / Q000032:analysis; D005033:Ethylmaleimide; D006801:Humans; D008970:Molecular Weight; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D014316:Tritium
D000596:Amino Acids; D001457:Anion Exchange Protein 1, Erythrocyte; D010446:Peptide Fragments; D014316:Tritium; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D010206:Papain; D005033:Ethylmaleimide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid