Proteolytic digestion of band 3 from bovine erythrocyte membranes in membrane-bound and solubilized states.
Bovine band 3 in membrane-bound and solubilized states was digested with chymotrypsin, trypsin, and papain. Bovine band 3 in red blood cells was fragmented by the proteases in a 5 mM NaH2PO4-Na2HPO4 buffer containing 0.3 M glucose, pH 8.0, but not in a 5 mM NaH2PO4-Na2HPO4 buffer containing 0.15 M NaCl, pH 8.0, in which human band 3 is cleaved by chymotrypsin and papain. When compared with the known data for human band 3, however, major fragments of bovine band 3 derived from intact cells, inside-out vesicles and unsealed ghosts were similar to those of human band 3, except that tryptic fragments were formed on the extracellular attack. The results suggest that bovine band 3 adopts a quite similar molecular arrangement in the membrane to in the human case. However, it was strongly suggested by molecular weight evaluation of fragments that the only detectable water-soluble 38,000-39,000 dalton fragment does not account for the entire hydrophilic pole of the band 3 molecule exposed in the cytoplasmic region of the membrane. When isolated band 3 was treated with the enzymes in a 2% solution of nonaethyleneglycol n-dodecyl ether, the major product was indistinguishable on sodium dodecyl sulfate-gel from the water-soluble fragment of the cytoplasmic domain origin of band 3. This fragment lost its resistance to further enzymatic degradation when treated with dimethylmaleic anhydride, thus band 3 oligomers were converted into their monomers. The chymotryptic 38,000 dalton water-soluble fragment obtained in nonaethyleneglycol n-dodecyl ether solution was a subfragment of a 50,000 dalton piece which was produced in a 2% solution of deoxycholate after chymotrypsin treatment of band 3.
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002918:Chymotrypsin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000648:ultrastructure*; D007700:Kinetics; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D012995:Solubility; D014357:Trypsin / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D010446:Peptide Fragments; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain