Identification of the hepatocyte Na+-dependent bile acid transport protein using monoclonal antibodies.
Monoclonal antibodies have been utilized to characterize the hepatocyte Na+-dependent bile acid transport system. Sinusoidal plasma membrane proteins in the 49-54-kDa range, which are thought to be components of this transport system, based on photo-affinity labeling and reconstitution studies, have been partially purified by affinity chromatography and utilized as an immunogen for the production of a panel of monoclonal antibodies (mAb). One of these mAbs, 25A-3, recognized both a 49- and a 54-kDa protein as assessed by immunoprecipitation. In addition, it was shown to protect the bile acid transport system from inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a dose-dependent manner. DIDS covalently labeled membrane proteins of 49 and 54 kDa, and this process could be significantly inhibited when performed in the presence of mAb 25A-3. Furthermore, the DIDS-labeled membrane proteins were immunoprecipitated by 25A-3. These results establish that one of these membrane components is the bile acid carrier protein. Another mAb (25D-1) which immunoprecipitated only a 49-kDa protein was shown to block the protective effect of 25A-3 on DIDS inhibition of bile acid transport. In addition both antibodies effected each other's binding capacity to hepatocytes and reacted with the same 49-kDa protein as established by sequential immunoprecipitation. Binding studies indicated that there are approximately 3.3 X 10(6) 49-kDa transport molecules/hepatocyte. These results firmly establish that the 49-kDa protein is the Na+-dependent hepatocyte bile acid transporter.
Ananthanarayanan
M
M
Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
von Dippe
P
P
Levy
D
D
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis*; D006913:Hydroxysteroid Dehydrogenases; D008099:Liver / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D010777:Photochemistry; D012964:Sodium / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000345:Affinity Labels; D000911:Antibodies, Monoclonal; 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)