Purification of a trypsin-insensitive fragment of spectrin from human erythrocyte membranes.
When spectrin is treated with trypsin, a series of polypeptide fragments is generated, One particular fragment having an approximate molecular weight of 80 000 constitutes 18% of the trypsin-digested mixture and is trypsin-insensitive. This fragment has been isolated and purified by gel filtration followed by ion-exchange chromatography. The molecular weight of the fragment, as seen from sedimentation equilibrium measurements and from gel electrophoresis, both in the presence and absence of detergent, is close to 80 000. There was no evidence of self-association under the conditions used. Changes in the specific rotation at 365 nm were used to detect temperature-dependent conformation changes in the fragment and to compare these changes with those in the intact spectrin molecule. The fragment undergoes temperature-dependent transitions centered at 46 and 58 degrees C, similar to those in intact spectrin (49 and 55 degrees C). Although the thermal transitions exhibited by intact spectrin are markedly salt-dependent, those shown by the fragment are not. ORD (optical rotary dispersion) measurements indicate 53% apparent alpha-helix in the fragment, compared to 68% in intact spectrin. Antibodies raised against the fragment cross-react only with band 1, the largest polypeptide of spectrin, indicating that the fragment is derived from band 1.
D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009904:Optical Rotation; D010446:Peptide Fragments / Q000097:blood* / Q000302:isolation & purification; D013049:Spectrin / Q000302:isolation & purification*; D014357:Trypsin
D007074:Immunoglobulin G; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D013049:Spectrin; D014357:Trypsin