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 0ยฐC in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ยฑ 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for โDIDS-Band 3โ complex, determined from the chloride transport experiments, was (34 ยฑ 3) nM and (80 ยฑ 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ยฑ 6) nM and (60 ยฑ 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 Hillโs coefficients were indistinguishable from 1 (within the limit error) both for control and EAC-treated ghosts. By using tritium-labeled DIDS, 4,4โ-diisothiocyanato-2,2โ-stilbenedisulfonate ([3H]DIDS), the association rate constant, k+1 (Mโ1sโ1), was measured. The mean values of (4.3 ยฑ 0.7) ร 105 Mโ1sโ1 for control and (2.7 ยฑ 0.7) ร 105 Mโ1sโ1 for EAC-treated ghosts were obtained. The mean values for KD, evaluated from [3H]DIDS binding measurements, were (37 ยฑ 9) nM and (90 ยฑ 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.