Red blood cell band 3. Lysine 539 and lysine 851 react with the same H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid) molecule.
The band 3 protein of the red blood cell membrane catalyzes anion exchange that is inhibited by the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid). There is one H2DIDS binding site per 95,000-Da band 3 polypeptide. The single bound H2DIDS molecule can react covalently with 2 different lysine residues. The 2 lysines that react covalently with H2DIDS have been localized directly by sequencing fragments of human band 3 from cells labeled with [3H]H2DIDS. The most rapid covalent reaction is with Lys-539, in agreement with site-directed mutagenesis studies. The slower reaction is with Lys-851, which is known to be the primary site of binding of another anion transport inhibitor, pyridoxal phosphate (Kawano et al., 1988). These results indicate that the protein is folded to bring these 2 residues into close enough proximity to react covalently with the same H2DIDS molecule. In addition to defining the residues that react with H2DIDS, these studies have also defined new in situ proteolytic cleavage sites in band 3.
Okubo
K
K
Department of Clinical Chemistry and Laboratory Medicine, Fukuoka University School of Medicine, Japan.
Kang
D
D
Hamasaki
N
N
Jennings
M L
ML
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D010450:Endopeptidases; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification
D001457:Anion Exchange Protein 1, Erythrocyte; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; C018564:dihydro-DIDS; D010450:Endopeptidases; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid