Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside.
Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)
D001457:Anion Exchange Protein 1, Erythrocyte / Q000302:isolation & purification*; D027321:Anion Transport Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002942:Circular Dichroism; D004910:Erythrocyte Membrane / Q000032:analysis*; D005960:Glucosides / Q000097:blood; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation
D001457:Anion Exchange Protein 1, Erythrocyte; D027321:Anion Transport Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D005960:Glucosides; C018619:octyl-beta-D-glucoside