Biochim Biophys Acta. 1992 Oct 19;1111(1):93-102. doi: 10.1016/0005-2736(92)90278-t.

Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

Biochimica et biophysica acta

W Kramer, S B Nicol, F Girbig, U Gutjahr, S Kowalewski, H Fasold

PMID: 1390867 DOI: 10.1016/0005-2736(92)90278-t

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.

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Substances

• Affinity Labels
• Bile Acids and Salts
• Carrier Proteins
• Membrane Glycoproteins
• bile acid binding proteins
• Sodium
• Hydroxysteroid Dehydrogenases
• AKR1C2 protein, human

MeSH terms

• Affinity Labels / metabolism
• Animals
• Bile Acids and Salts / metabolism
• Biological Transport / drug effects
• Carrier Proteins / analysis
• Hydroxysteroid Dehydrogenases*
• Ileum / metabolism
• In Vitro Techniques
• Male
• Membrane Glycoproteins*
• Microvilli / metabolism
• Rabbits
• Sodium / pharmacology
• Structure-Activity Relationship
• Substrate Specificity

Publication Types

• Journal Article

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