title: Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.
abstract: The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.
authors.affiliation: Hoechst Aktiengesellschaft, Frankfurt am Main, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Kramer
authors.forename: S B
authors.initials: SB
authors.lastname: Nicol
authors.forename: F
authors.initials: F
authors.lastname: Girbig
authors.forename: U
authors.initials: U
authors.lastname: Gutjahr
authors.forename: S
authors.initials: S
authors.lastname: Kowalewski
authors.forename: H
authors.initials: H
authors.lastname: Fasold
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000032:analysis; D006913:Hydroxysteroid Dehydrogenases; D007082:Ileum / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008562:Membrane Glycoproteins; D008871:Microvilli / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
chemical_list: D000345:Affinity Labels; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; D012964:Sodium; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)