Reversible DIDS binding to band 3 protein in human erythrocyte membranes.
Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.
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Department of Physics, Technical University, Zielona Góra, Poland.
Janas
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D013816:Thermodynamics; D014316:Tritium
D001457:Anion Exchange Protein 1, Erythrocyte; D014316:Tritium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid