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

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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 ([3H]DIDS), the association rate constant, k+1 (M−1s−1), was measured. The mean values of (4.3 ± 0.7) × 105 M−1s−1 for control and (2.7 ± 0.7) × 105 M−1s−1 for EAC-treated ghosts were obtained. The mean values for KD, evaluated from [3H]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.

Abbreviations

DIDS:

4,4’-diisothiocyanato-2,2’-stilbenedisulfonate

DNDS:

4,4’-dinitrostilbene-2,2’-disulfonate

EAC:

1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide

H2DIDS:

4,4’-diisothiocyanodihydro-2,2’-stilbenedisulfonate

TEE:

tyrosine ethyl ester

WRK, Woodward’s reagent K:

N-ethyl-5-phenylisoxazolium 3’-sulfonate

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Correspondence to Teresa Janas.

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Key words

  • Band 3
  • Carbodiimide
  • Dissociation constant
  • Erythrocyte membrane
  • Stilbenedisulfonate