title: Purification and properties of human erythrocyte band 4.2. Association with the cytoplasmic domain of band 3.
abstract: We have purified the human erythrocyte membrane protein band 4.2 to greater than 85% homogeneity. The protein was extracted from spectrin-actin-depleted inside-out vesicles in a pH 11 medium and purified by gel filtration in the presence of 1 M KI. The purified protein was heterogeneous and had an average S20,w of 5.5 and an average Stokes radius of 82 A. By electron microscopy, the protein appeared heterogeneous in size and shape, having a diameter ranging from 80 to 150 A. The protein bound saturably to band 4.2-depleted red cell inside-out vesicles, and the binding exhibited a concave Scatchard plot. Binding was reduced greater than 90% by proteolytic digestion of membranes. Digestion studies suggested that there are two classes of binding sites for band 4.2 on the cytoplasmic aspect of red cell membranes, one of which is likely to be band 3. The purified 43-kDa cytoplasmic domain of band 3 competed for band 4.2 binding to red cell membranes and could completely abolish binding when added at a concentration of greater than 200 micrograms/ml. The purification of band 4.2 and the characterization of its association with red cell membranes should facilitate the discovery of the function of this major red cell membrane protein.
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis*; D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000032:analysis; D003598:Cytoskeletal Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins; D008722:Methods; D008854:Microscopy, Electron
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; C044791:erythrocyte membrane band 4.2 protein