AJP - Cell Physiology, Vol 259, Issue 3 C439-C449, Copyright © 1990 by American Physiological Society
Fluorescent stilbene (BADS) binding proteins in anion-transporting epithelia
S. F. Pearce and J. A. Zadunaisky
Department of Physiology and Biophysics, New York University School of Medicine, New York 10016.
Chloride transport occurs at the interface between the internal and
external environments of a cell where chloride uptake or efflux is
regulated through a variety of mechanisms that involve cotransport of
cations, exchange mechanism with anions, or movement through channels. One
of these mechanisms, a chloride-bicarbonate exchange found in the human red
blood cell, is well characterized and is mediated by a protein commonly
known as band 3. To ascertain the presence of this or other mechanisms in
epithelia, the sensitivity of epithelial membranes toward stilbenes was
examined. Structure function activities of stilbene derivatives with red
cell ghosts show that stilbene molecules block anion transport sites. One
of these stilbenes, 4-benzamido-4'-aminostilbene-2-2'-disulfonic acid
(BADS), chosen for its property of enhanced fluorescence on binding to
hydrophobic sites, was used as a probe to examine the presence or absence
of similar sites on epithelial membranes. With the use of nonlinear curve
fitting, a single class of sites was found for BADS in the rat kidney
cortex (1.6 microM), rat kidney medulla (2.1 microM), rat small intestine
(2.2 microM), rat pancreatic islets (5.8 microM), frog cornea (4.3 microM),
and shark rectal gland (1.5 microM). In the presence of chloride, the
affinity for BADS decreased in all tissues except the frog corneal
epithelium where it remained unchanged. The binding of BADS could be
displaced by loop diuretics (furosemide, bumetanide, and piretanide) and
thiocyanate anion in the kidney, intestine, and shark rectal gland; 50%
displacement occurred at approximately 40 microM concentrations for
furosemide with an order of magnitude less for bumetanide. The
near-millimolar concentrations required for the displacement of BADS by
loop diuretics indicate that this effect is nonspecific. However, the
effect of chloride, thiocyanate, and loop diuretics on the binding of BADS
indicates that BADS possibly interacts with an anion site.
