In situ cross-linking of human erythrocyte band 3 by bis(sulfosuccinimidyl)suberate. Evidence for ligand modulation of two alternate quaternary forms: covalent band 3 dimers and noncovalent tetramers formed by the association of two covalent dimers.
Treatment of intact human erythrocytes with bis(sulfosuccinimidyl)suberate converted band 3 to two species with lower electrophoretic mobility in sodium dodecyl sulfate (SDS). The presence of the noncovalent anion transport inhibitor, 4,4'-dinitrostilbene-2,2'-disulfonate, promoted the lowest mobility form, while a closely related analogue, 4,4'-diisothiocyano-2,2'-stilbenedisulfonate, did not. Ferguson analysis of the electrophoretic behavior of the two slowly migrating bands strongly suggested that they represented dimers and tetramers of band 3. Increasing the temperature of the SDS solution to greater than 60 degrees C quantitatively converted the tetrameric species to the dimeric form. We conclude that band 3 can be intermonomerically cross-linked by bis(sulfosuccinimidyl)suberate as covalent dimers within two alternate quaternary forms in a manner modulated by the ligand occupying the intramonomeric stilbenedisulfonate site. In one form, band 3 covalent dimers are noncovalently associated as a SDS-resistant tetramer, while in the other form, covalent dimers are not so associated. There is no obvious relationship between ligand stereochemistry and the resulting quaternary form, suggesting that the two forms reflect alternate allosterically modulated porter quaternary structures. The significance of these two quaternary states to the transport or the ankyrin binding functions of band 3 is unknown.
Veterans Administration Medical Center, Omaha, Nebraska.
J M
JM
Salhany
R L
RL
Sloan
K A
KA
Cordes
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D013267:Stilbenes / Q000494:pharmacology; D013388:Succinimides / Q000494:pharmacology*; D013816:Thermodynamics
D001457:Anion Exchange Protein 1, Erythrocyte; D003432:Cross-Linking Reagents; D008024:Ligands; D046911:Macromolecular Substances; D013267:Stilbenes; D013388:Succinimides; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; C035760:bis(sulfosuccinimidyl)suberate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid