TY - JOUR
T1 - Kinetic mechanism of DIDS binding to band 3 (AE1) in human erythrocyte membranes
AU - Salhany, James M.
AU - Schopfer, Lawrence M.
N1 - Funding Information:
We thank Karen Cordes and Renee Sloan for assistance. This work was supported by the Medical Research Service of the Veterans Administration.
PY - 2001
Y1 - 2001
N2 - Stilbenedisulfonates (S) are used widely in cell biology as competitive inhibitors of anion exchange, but the mechanism of competition is not resolved. Resolution requires understanding the detailed steps in the reaction of stilbenedisulfonates with various anion-exchange proteins. Studies on the reversible binding of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) and H2DIDS (4,4′-diisothiocyanatodihydro-2,2′-stilbenedisulfonate) to erythrocyte band 3 (B) have shown biphasic kinetic time courses at 25°C. Yet, results for the reversible binding of DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) are controversial. One recent report has shown monophasic kinetics, in experiments performed at 0°C, and at a single, very low concentration of DIDS (0.1 μM). Studies are presented which attempt to reconcile these recent findings with the other kinetic data in the literature. We measure the kinetics of DIDS reversible binding to band 3, over a wide DIDS concentration range. In addition, the time course for DIDS binding to band 3 at 0°C is compared with that at 25°C. The results show biphasic binding kinetics at both 0 and 25°C, and they are consistent with expectations for a two-step binding mechanism (S + B ⇄ SB ⇄ SB*). Furthermore, computer-assisted model simulation studies reveal that monophasic DIDS binding kinetics are generated by a two-step mechanism, when calculations are performed at 0.1 μM DIDS and 0°C. Under these conditions the initial binding step in the two-step reaction becomes rate limiting. We conclude that the two-step binding mechanism best describes stilbenedisulfonate binding to band 3 and that the observation of monophasic kinetics at low concentrations of DIDS, while valid, is not mechanistically discriminating, since both one-step and two-step mechanisms can yield the same result.
AB - Stilbenedisulfonates (S) are used widely in cell biology as competitive inhibitors of anion exchange, but the mechanism of competition is not resolved. Resolution requires understanding the detailed steps in the reaction of stilbenedisulfonates with various anion-exchange proteins. Studies on the reversible binding of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) and H2DIDS (4,4′-diisothiocyanatodihydro-2,2′-stilbenedisulfonate) to erythrocyte band 3 (B) have shown biphasic kinetic time courses at 25°C. Yet, results for the reversible binding of DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) are controversial. One recent report has shown monophasic kinetics, in experiments performed at 0°C, and at a single, very low concentration of DIDS (0.1 μM). Studies are presented which attempt to reconcile these recent findings with the other kinetic data in the literature. We measure the kinetics of DIDS reversible binding to band 3, over a wide DIDS concentration range. In addition, the time course for DIDS binding to band 3 at 0°C is compared with that at 25°C. The results show biphasic binding kinetics at both 0 and 25°C, and they are consistent with expectations for a two-step binding mechanism (S + B ⇄ SB ⇄ SB*). Furthermore, computer-assisted model simulation studies reveal that monophasic DIDS binding kinetics are generated by a two-step mechanism, when calculations are performed at 0.1 μM DIDS and 0°C. Under these conditions the initial binding step in the two-step reaction becomes rate limiting. We conclude that the two-step binding mechanism best describes stilbenedisulfonate binding to band 3 and that the observation of monophasic kinetics at low concentrations of DIDS, while valid, is not mechanistically discriminating, since both one-step and two-step mechanisms can yield the same result.
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U2 - 10.1006/bcmd.2001.0458
DO - 10.1006/bcmd.2001.0458
M3 - Article
C2 - 11783947
AN - SCOPUS:0035207098
SN - 1079-9796
VL - 27
SP - 844
EP - 849
JO - Blood Cells, Molecules, and Diseases
JF - Blood Cells, Molecules, and Diseases
IS - 5
ER -