Major proteolytic fragments of the murine band 3 protein as obtained after in situ proteolysis.
Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites. Exposure of the intact murine red cell to chymotrypsin leads to the formation of two fragments of 67 kDa and 41 kDa, which are equivalent to the 60 kDa and the 35 kDa fragments of the human band 3. Internal trypsin cleaves the chymotryptic 67 kDa fragment while the 41 kDa fragment appears essentially unaffected. The 67 kDa fragment is first degraded to 64 kDa, then further to 22 kDa and finally to 19 kDa. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) combines with murine band 3 protein as it does with human band 3. Anion transport is maximally inhibited when 5.10(5) H2DIDS molecules per cell are bound to band 3. As in the human red cell, after exposure to high pH (9.0-9.5) of the H2DIDS-labeled, chymotryptically cleaved band 3 intramolecular cross-linking takes place. This joins the 67 and 41 kDa chymotryptic pieces together to form a peptide of the original molecular mass of band 3 of 108 kDa. If cross-linking is performed after additional tryptic cleavage, the 19 and 22 kDa pieces join together with 41 kDa pieces to form overlapping bands that cover the molecular weight range from 60 to 63 kDa.
Raida
M
M
Max-Planck-Institut für Biophysik, Frankfurt am Main, F.R.G.
Wendel
J
J
Kojro
E
E
Fahrenholz
F
F
Fasold
H
H
Legrum
B
B
Passow
H
H
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence / Q000187:drug effects; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002918:Chymotrypsin / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D008051:Lipid Bilayers; D010446:Peptide Fragments; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D002918:Chymotrypsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid