Identification of taurocholate binding sites in ileal plasma membrane.
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)
Hepatobiliary Research Center, University of Colorado School of Medicine, Denver.
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Simon
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Sutherland
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Sutherland
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases; D007082:Ileum / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins; D008871:Microvilli / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D009994:Osmolar Concentration; D051381:Rats; D011919:Rats, Inbred Strains; D013656:Taurocholic Acid / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; D013656:Taurocholic Acid; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)