Catabolism of the anion transport protein in human erythrocytes.
We identified the catabolic products of protein 3 in human erythrocytes. Protein 3, the major protein of the erythrocyte membrane, functions in anion transport and reacts covalently with tritiated 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid ([3H]DIDS), a very selective inhibitor of anion transport. In this study, [3H]DIDS was used to label protein 3 in the membranes of normal cells and those from a donor heterozygous for a variant of protein 3, defined by its elongated amino-terminal end. Both types of cells contained [3H]DIDS-labeled peptides other than protein 3. A protein fragment of 60K molecular weight was found in normal cells, whereas both 60K and 63K fragments were identified in cells from the heterozygote. These peptides are identical with those generated by treatment of intact erythrocytes with Pronase or chymotrypsin. A polyclonal rabbit antibody specific for the purified 60K fragment of protein 3 was used to detect this protein and its products in the erythrocyte membrane. Autoradiographs of membrane peptides that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and allowed to react with the monospecific antibody showed, in addition to protein 3, a 60K fragment and fragments in the 40K region and in the 20-30K region. Cells containing the protein 3 variant yielded two fragments showing a 3K difference in molecular weight in all three regions, demonstrating that degradation of protein 3 is identical in normal erythrocytes and those heterozygous for the variant. This observation also confirms the common derivation of the fragments from protein 3.(ABSTRACT TRUNCATED AT 250 WORDS)
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism; D027321:Anion Transport Proteins; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D012016:Reference Values
D001457:Anion Exchange Protein 1, Erythrocyte; D027321:Anion Transport Proteins; D002352:Carrier Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid