AJP - Gastrointestinal and Liver Physiology, Vol 259, Issue 3 394-G401, Copyright © 1990 by American Physiological Society
Identification of taurocholate binding sites in ileal plasma membrane
F. R. Simon, J. Sutherland and E. Sutherland
Hepatobiliary Research Center, University of Colorado School of Medicine, Denver.
Intestinal absorption of bile salts occurs by passive processes throughout
the length of the small intestine, whereas active carrier-mediated uptake
is localized to the ileum. Although previous studies have extensively
characterized brush-border transport of bile acids, their extrusion across
the basolateral membrane is less well understood. Because previous reports
had failed to show specific bile acid binding sites except with the use of
photolabeled bile salt derivatives, we sought to identify and characterize
the binding parameters of the physiological bile salt taurocholate in ileal
and jejunal plasma membrane subfractions. Brush-border membrane (BBM) and
basolateral membrane (BLM) fractions were rapidly and simultaneously
isolated from the small intestinal mucosa. BBM fractions were isolated with
enrichments of 50- to 54-fold for leucine aminopeptidase, whereas the
basolateral membrane enrichment of Na(+)-K(+)-ATPase, its specific marker
enzyme, was 22- to 25-fold. Contamination from intracellular organelles was
minimal. Binding of [14C]taurocholate was demonstrated in both jejunal as
well as ileal plasma membrane fractions. However, only ileal binding
demonstrated saturation, reversibility, and susceptibility to proteolytic
enzymes. [14C]taurocholate binding to BBM fractions also showed competition
with bile acids but was not altered by pH or alkylating agents. In
contrast, binding of taurocholate to the basolateral membrane showed
optimal pH between 6.5 and 7.5 and was inhibited by thiol and alkylating
agents. Kinetic analysis of specific ileal BBM and BLM binding showed the
parameters for BBM as 288 +/- 70 microM and 2.4 +/- 0.6 nmol/mg protein and
for BLM as 6.6 +/- 0.7 microM and 0.56 +/- 0.01 nmol/mg protein for
dissociation constant and maximum binding capacity, respectively.(ABSTRACT
TRUNCATED AT 250 WORDS)
