Fragmentation of the 95,000-dalton transmembrane polypeptide in human erythrocyte membranes.
The 95,000-dalton polypeptide in human red blood cell membranes constitutes about 25% of the membrane protein. Previous labeling studies have shown that different regions of this polypeptide are exposed to the inside and outside of the cell and have suggested a role for the protein in anion exchange across the membrane. This polypeptide has been fragmented by chymotrypsin digestion of intact red cells and by treatment of purified polypeptide with 2-nitro-5-thiocyanobenzoic acid, hydroxylamine, and N-bromosuccinimide. The sites of cleavage by each of these reagents have been located relative to the NH-2 and COOH-terminals of the intact 95,000-dalton polypeptide. Polypeptide obtained from cells labeled with 1-isothiocyanate-4-benzene [35S]sulfonic acid (an inhibitor of anion transport), 125I and lactoperoxidase, or 32P has been similarly fragmented and these labels have been assigned to specific regions of the polypeptide. There are at least two sites of phosphorylation of the polypeptide; the major sites lies within 10,000 daltons of the NH2-terminal requiring that this portion of the polypeptide lie inside the cell. Sites of chymotrypsin cleavage and 125I and lactoperoxidase labeling are in a 7,000-dalton region toward the COOH-terminal of the polypeptide; this region must lie outside the cell. Between these two regions the polypeptide must traverse the lipid bilayer an odd number of times. 1-Isothiocyanate-4-benzenesulfonic acid also labels the protein near the site of chymotrypsin cleavage.
D001798:Blood Proteins / Q000302:isolation & purification; D002268:Carboxypeptidases; D002462:Cell Membrane / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000648:ultrastructure*; D006801:Humans; D008055:Lipids / Q000097:blood*; D008567:Membranes, Artificial; D008954:Models, Biological; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000097:blood*
D001798:Blood Proteins; D008055:Lipids; D008567:Membranes, Artificial; D010446:Peptide Fragments; D010455:Peptides; D002268:Carboxypeptidases