Monoclonal antibodies against human erythrocyte band 3 protein. Localization of proteolytic cleavage sites and stilbenedisulfonate-binding lysine residues.
Monoclonal antibodies against the membrane domain of human red blood cell band 3 protein have been prepared and used in topographical studies of the arrangement of the polypeptide in the membrane. One of the antibodies binds to a site near the N terminus of the membrane domain; another binds to a site near the C terminus. The latter has been used to localize a site of intracellular trypsin digestion. The cleavage site, in human band 3, corresponds to Lys-761 in mouse band 3; the site is 168 residues from the C terminus of the protein. This is the first intracellular site in the membrane domain (other than the N terminus) that has been localized in the primary structure. The antibody that binds to the N-terminal portion of the membrane domain has been used to identify a new S-cyanylation cleavage site about 7,000 daltons from the C terminus. Proteolysis/cross-linking experiments with the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) reveal that one end of the H2DIDS reacts covalently with a lysine residue that is between about 70 and 168 residues from the C terminus of band 3. In addition to placing restrictions on the location of the H2DIDS-binding lysine, these studies provide direct evidence that the C-terminal 28,000-dalton papain fragment crosses the membrane at least three times. With previous data on the remainder of the membrane domain, there is now direct evidence that the band 3 polypeptide crosses the membrane at least eight times.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008239:Lysine; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013267:Stilbenes; D014357:Trypsin
D001457:Anion Exchange Protein 1, Erythrocyte; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D014357:Trypsin; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid