title: Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes.
abstract: Noncovalent DIDS binding to Band 3 (AE1) protein in human erythrocyte membranes, modified by non-penetrating, water soluble 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC), was studied at 0°C in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ± 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for "DIDS-Band 3" complex, determined from the chloride transport experiments, was (34 ± 3) nM and (80 ± 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ± 6) nM and (60 ± 8) nM in control and EAC-treated ghosts, respectively. The reduced affinity for DIDS reversible binding was not a result of negative cooperativity of DIDS binding sites of Band 3 oligomer since Hill's coefficients were indistinguishable from 1 (within the limit error) both for control and EAC-treated ghosts. By using tritium-labeled DIDS, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate ([(3)H]DIDS), the association rate constant, k(+1) (M(-1)s(-1)), was measured. The mean values of (4.3 ± 0.7) × 10(5) M(-1)s(-1) for control and (2.7 ± 0.7) × 10(5) M(-1)s(-1) for EAC-treated ghosts were obtained. The mean values for K(D), evaluated from [(3)H]DIDS binding measurements, were (37 ± 9) nM and (90 ± 21) nM for control and EAC-modified ghosts, respectively. The results demonstrate that EAC modification of AE1 reduces about 2-fold the affinity of AE1 for DIDS. It is suggested that half of the subunits are modified near the transport site by EAC.
authors: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, USA. teresa.janas@colorado.edu
authors: Teresa
authors: T
authors: Janas
authors: Tadeusz
authors: T
authors: Janas
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport; D007700:Kinetics; D011485:Protein Binding; D014316:Tritium / Q000737:chemistry
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D002234:Carbodiimides; D002712:Chlorides; D014316:Tritium; C044568:1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid