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We recently purified two closely related 33 kDa proteins from rat hepatic cytosol, designated bile acid binder I and II, which selectively bind bile acids with comparable affinity as glutathione S‐transferase B. This work has now been extended to human liver in which we have identified a similar cytosolic binding activity in the 30–40 kDa fraction from gel filtration. Subsequent chromatofocusing and hydroxyapatite chromatography resulted in the isolation of a homogenous monomeric protein of 36 kDa. The binding affinity of this protein for lithocholate using the displacement of 1‐anilino‐8‐naphthalenesulfonate (ANS) was 0.1 μM, whereas human hepatic glutathione S‐transferases purified from glutathione affinity chromatography demonstrated no competitive displacement of ANS.

We recently purified two closely related 33 kDa proteins from rat hepatic cytosol, designated bile acid binder I and II, which selectively bind bile acids with comparable affinity as glutathione S-transferase B. This work has now been extended to human liver in which we have identified a similar cytosolic binding activity in the 30–40 kDa fraction from gel filtration. Subsequent chromatofocusing and hydroxyapatite chromatography resulted in the isolation of a homogenous monomeric protein of 36 kDa. The binding affinity of this protein for lithocholate using the displacement of 1-anilino-8-naphthalenesulfonate (ANS) was 0.1 μM, whereas human hepatic glutathione S-transferases purified from glutathione affinity chromatography demonstrated no competitive displacement of ANS.

In the gel filtration of 100,000 g rat liver supernatant, four major gluta-thione S-transferase activities, S-aryl-, S-epoxide-, S-aralkyl-, and S-alkyltransferase, were identified as having an elution volume identical to that of fractions exhibiting either glutathione or sulfobromophthalein sodium binding. The organic anions, sulfobromophthalein sodium, indocyanine green, and bilirubin, were found to be competitive inhibitors of the four glutathione S-transferase activities. These findings indicate that the glutathione S-transferases bind organic anions and, as a group, have a similar molecular weight to a known organic anion-binding protein. It is proposed that these enzymes also serve nonenzymically as a group of binding proteins in the hepatic cytoplasmic transport of organic anions.

The relationship between biliary secretion of bilirubin and glutathione was investigated by infusing bilirubin solution (200 nmol/min/100 g body wt) into Sprague-Dawley rats and measuring bilirubin and glutathione in the bile. Hepatic glutathione level, when modified between the range of 1–10 μmol/g liver wt, did not affect biliary maximal secretory rate (Tm) of bilirubin (80 nmol/min/100 g body wt). However, when biliary secretion of bilirubin exceeded 10 mM or 30 nmol/min/100 g body wt, biliary secretion of glutathione was markedly impaired while the bile flow remained relatively constant. Thus, bilirubin impaired the biliary secretion of glutathione selectively compared to its effect on bile formation. The results indicate that the mechanisms of biliary secretion of the two physiological substances, bilirubin and glutathione, are closely related.