The interaction of human erythrocyte Band 3 with cytoskeletal components.
Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of 35SO2-4 was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 N NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 N NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D000838:Anions / Q000097:blood; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D055598:Chemical Phenomena; D002621:Chemistry; D003599:Cytoskeleton / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010455:Peptides / Q000097:blood; D011485:Protein Binding / Q000187:drug effects; D013267:Stilbenes / Q000494:pharmacology*
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D010455:Peptides; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid