Isolation and characterization of the canalicular membrane bile acid transport protein of rat liver.
The aim of this study was to isolate the Na(+)-independent bile acid transporter from rat canalicular plasma membranes by affinity chromatography. The affinity matrix used consisted of lysylcholic acid covalently linked to CH-Sepharose 4B, resulting in an anionic ligand essentially identical to glycocholic acid. The protein fraction, adsorbed and eluted from the affinity column, was markedly enriched in a 100-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared with the total membrane and membrane extract. The 100-kDa band, further purified by preparative SDS-PAGE, was electroeluted from excised gel fragments and used as an immunogen for antibody production in rabbits. The immune serum, but not preimmune serum, specifically recognized a single, 100-kDa polypeptide on one- and two-dimensional immunoblots of canalicular membranes. In contrast, no reactivity was observed with proteins in liver basolateral or ileal brush-border membranes. The 125I-labeled protein was immunoprecipitated from membrane extracts solubilized in NP-40 and was found to migrate with a pI of 5.3 on two-dimensional electrophoresis. The apparent molecular weight of the protein was reduced by 50% after deglycosylation with N-glycanase. The 100-kDa protein was localized specifically and exclusively by immunocytochemical methods to the bile canalicular domain of the hepatocyte plasma membrane. Moreover, the immunoglobin G fraction prepared from the antiserum significantly inhibited taurocholate transport by canalicular membrane vesicles and decreased the covalent labeling of the 100-kDa protein by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus the isolation of a single 100-kDa protein by bile acid-affinity chromatography, as well as the inhibitory effects of antibodies directed against this polypeptide, provide further support for its role in the canalicular transport of bile acids.
Sippel
C J
CJ
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510.
Ananthanarayanan
M
M
Suchy
F J
FJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006913:Hydroxysteroid Dehydrogenases; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D000596:Amino Acids; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)