AJP - Cell Physiology, Vol 264, Issue 5 C1155-C1164, Copyright © 1993 by American Physiological Society
Lys-430, site of irreversible inhibition of band 3 Cl- flux by eosin-5-maleimide, is not at the transport site
S. Q. Liu and P. A. Knauf
Department of Biophysics, University of Rochester Medical Center, New York 14642.
Although eosin-5-maleimide (EM) covalently labels band 3 and has been
thought to react at the external-facing anion transport site, EM reversibly
inhibits Cl- exchange at 0 degrees C in a noncompetitive fashion,
indicating that under these conditions it does not bind to the transport
site [Knauf, P.A., N.M. Strong, J. Penikas, R.B. Wheeler, Jr., and S.J.
Liu. Am. J. Physiol. 264 (Cell Physiol. 33): C1144-C1154 1993]. To see
whether or not the covalent labeling by EM takes place at the same
noncompetitive site as the reversible binding, we examined the dependence
of reaction rate on EM concentration. The reaction rate saturates with
increasing EM concentration, indicating that reversible binding precedes
covalent reaction and that EM therefore acts as an affinity label. A more
complex model in which reversible binding prevents a bimolecular reaction
at a different site cannot, however, be ruled out. Cl- gradients across the
membrane affect EM reversible binding in a manner suggesting that EM binds
preferentially to the Eo form of band 3, with the transport site unloaded
and facing outward. Thus EM binds to and probably reacts covalently with a
site that is different from the transport site, but whose conformation is
affected by the orientation of the transport site. Lysine-430, the amino
acid residue which is covalently labeled by EM (4), may be near the
transport site but does not seem to be directly involved in the binding of
transported substrates such as chloride. EM binding to one band 3 monomer
decreases the reactivity of the adjacent monomer but does not decrease the
affinity constant of the reversible binding step that precedes covalent
reaction. Although a small fraction (approximately 1%) of band 3 monomers
fail to react with EM, EM nearly completely inhibits transport in those
monomers with which it reacts.
