Isolation and characterization of the putative canalicular bile salt transport system of rat liver.
Through labeling with the sodium salt of the photolabile bile salt derivative (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)- 2-aminoethanesulfonic acid, a bile salt-binding polypeptide with an apparent molecular weight of 100,000 was identified in isolated canalicular but not basolateral (sinusoidal) rat liver plasma membranes. This labeled polypeptide was isolated from octyl glucoside-solubilized canalicular membranes by DEAE-cellulose and subsequent wheat germ lectin Sepharose chromatography. The purified protein still contained covalently incorporated radioactive bile salt derivative and exhibited a single band with an apparent molecular weight of 100,000 on sodium dodecyl sulfate-gels. Antibodies were raised in rabbits and their monospecificity toward this canalicular polypeptide demonstrated by immunoblot analysis. No cross-reactivity was found with basolateral membrane proteins. The antibodies inhibited taurocholate uptake into isolated canalicular but not basolateral membrane vesicles. In addition, the antibodies also decreased efflux of taurocholate from canalicular vesicles. If the canalicular bile salt-binding polypeptide was immunoprecipitated from Triton X-100-solubilized canalicular membranes and subsequently deglycosylated with trifluoromethanesulfonic acid, the apparent molecular weight was decreased from 100,000 to 48,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These studies confirm previous results in intact liver tissue and strongly indicate that a canalicular specific glycoprotein with an apparent molecular weight of 100,000 is directly involved in canalicular excretion of bile salts.
D000345:Affinity Labels; D000818:Animals; D001391:Azo Compounds / Q000378:metabolism; D001648:Bile Canaliculi / Q000032:analysis*; D001653:Bile Ducts, Intrahepatic / Q000032:analysis*; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification*; D002462:Cell Membrane / Q000032:analysis; D000939:Epitopes / Q000276:immunology; D006913:Hydroxysteroid Dehydrogenases; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay; D008099:Liver / Q000032:analysis*; D008297:Male; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D010777:Photochemistry; D051381:Rats; D011919:Rats, Inbred Strains; D013656:Taurocholic Acid / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D000345:Affinity Labels; D001391:Azo Compounds; D002352:Carrier Proteins; D000939:Epitopes; D006913:Hydroxysteroid Dehydrogenases; D007106:Immune Sera; D008562:Membrane Glycoproteins; D013656:Taurocholic Acid; C037643:bile acid binding proteins; C037641:(7,7-azo-3,12-dihydroxy-5-cholan-24-oyl)-2-aminoethanesulfonic acid; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)