AJP - Cell Physiology, Vol 265, Issue 2 C521-C532, Copyright © 1993 by American Physiological Society
WW-781, a potent reversible inhibitor of red cell Cl- flux, binds to band 3 by a two-step mechanism
N. M. Raha, L. J. Spinelli and P. A. Knauf
Department of Biophysics, University of Rochester Medical Center, New York 14642.
WW-781 ([3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbit uric
acid]-pentamethine oxonol), a fluorescent dye that has been used for
measuring membrane potentials by optical methods, inhibits human red blood
cell Cl- exchange, which is mediated by the membrane protein known as band
3 or capnophorin. The inhibition is slowly reversible upon removal of
WW-781 from the medium, with a half time of approximately 4.7 min in 150 mM
Cl- medium at 0 degrees C. The mechanism of inhibition by WW-781 involves a
two-step binding reaction. WW-781 binds rapidly to band 3 to form an
initial complex, which can also rapidly dissociate. Formation of this
initial complex is followed by the much slower formation of a second
complex (with a rate constant of approximately 1.1 min-1), probably
involving a protein conformational change, through which WW-781 is more
tightly bound to band 3. At low concentrations, WW-781 inhibits Cl-
exchange with a stoichiometry of 1 WW-781 molecule per band 3 monomer,
suggesting that under these conditions the binding of WW-781 is highly
selective for the band 3 protein.
