Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes

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 0A degrees 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) x 10(5) M(-1)s(-1) for control and (2.7 +/- 0.7) x 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.