Reconstitution of the immunopurified 49-kDa sodium-dependent bile acid transport protein derived from hepatocyte sinusoidal plasma membranes.
Reconstitution, using phosphatidylcholine liposomes in conjugation with immunological purification procedures, has been used to establish directly the identity of the hepatocyte Na(+)-dependent bile acid transport protein. Octyl glucoside-solubilized sinusoidal plasma membranes were shown to form proteoliposomes exhibiting taurocholate transport properties which were similar to those of plasma membrane vesicles, namely, Na(+)-dependence and marked inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and by taurochenodeoxycholate. Proteoliposomes formed from plasma membrane proteins depleted of the putative 49-kDa bile acid transport protein by immunoprecipitation with monoclonal antibody 25D-1, which specifically recognizes this protein (Ananthanarayanan, M., von Dippe, P., and Levy, D. (1988) J. Biol. Chem. 263, 8338-8343), showed a 94% reduction in mediated transport capacity. Proteoliposomes containing total membrane protein also demonstrated Na(+)-dependent alanine transport. The addition of taurochenodeoxycholate or the removal of the 49-kDa protein by monoclonal antibody 25D-1 immunoprecipitation had no effect on the uptake of alanine, thus confirming the specificity of these procedures. When only the immunoprecipitated 48-kDa protein was used in the reconstitution system, a 2200% increase of taurocholate uptake was observed. These results definitively establish that this 49-kDa sinusoidal membrane protein is the sole essential component of the Na(+)-dependent bile acid transport system.
von Dippe
P
P
Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles 90033.
Levy
D
D
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology*; D013656:Taurocholic Acid / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D006913:Hydroxysteroid Dehydrogenases; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011510:Proteolipids; D012964:Sodium; D013656:Taurocholic Acid; C037643:bile acid binding proteins; C020485:proteoliposomes; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)