| Band 3, the anion channel protein of the human erythrocyte, is the major transmembrane glycoprotein of the erythrocyte membrane. On SDS* polyacrylamide gels the protein is distributed in a broad 88,000-98,000 dalton band. Previous investigations of the protein's structure by partial proteolysis have defined peptide fragments that span the amino terminal and middle regions of the molecule. These fragments define an amino terminal cytoplasmic domain and an adjacent membrane spanning domain of the Band 3 molecule. The fragments containing these domains appear as discrete bands by SDS gel electrophoresis and have molecular weights of 40,000 and 19,000.;The carboxyl terminal peptide fragment of the Band 3 molecule has a characteristic peptide map. A fragment containing this region is generated by digestion with chymotrypsin at the external face of intact erythrocytes. This fragment has a site accessible to lactoperoxidase catalyzed iodination when the enzyme is restricted to the internal face of the erythrocyte membrane. On this basis it is postulated that the fragment spans the bilayer and the carboxyl terminal domain of the protein thus represents a second membrane-spanning domain of the molecule.;The carboxyl terminal peptide fragment of Band 3 also appears as a broad band of 34,000-45,000 daltons apparent weight when analyzed by SDS polyacrylamide gel electrophoresis. Subpopulations of this peptide of differing electrophoretic mobility nonetheless have identical peptide maps when this segment is further digested by either trypsin or chymotrypsin. Similarly subpopulations of Band 3 of differing electrophoretic mobility also have identical peptide maps when the molecule is fragmented by either trypsin or chymotrypsin. The peptide maps are shown to represent the complete Band 3 molecule and the total material in the broad Band 3 gel band. Therefore Band 3 and its proteolytic fragments are each single peptides with homogeneous peptide backbones, despite their heterogeneous appearance on SDS polyacrylamide gels. This conclusion is supported by the finding of a unique amino terminal tetrapeptide sequence for one Band 3 peptide with a heterogeneous appearance on SDS polyacrylamide gels. The apparent heterogeneity of Band 3 seen on gel electrophoresis is associated with its carboxyl terminal region and reflects either variability of glycosylation of this region or variability of SDS binding to this region, or both.;This study has employed the technique of two-dimensional peptide mapping to reinvestigate the relationship of Band 3 to its proteolytic fragments, and of Band 3 to the Band 3 subpopulations defined by their differing electrophoretic mobility. |
