Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases.
Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism*; D006913:Hydroxysteroid Dehydrogenases; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D000596:Amino Acids; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D005982:Glutathione Transferase; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)