A monoclonal antibody, PKlB, directed against rat liver bile acid dfotransferase was used for the purification and characterization of the enzyme. Incubation of rat liver supernatant with the antibody followed by immunoprecipitation with Staphylococcus ~ureus cells demonstrated that PKlB reacted with 90% of the enzymatic activity present in the liver supernatant from female rats and 40 to 50% of the activity in male liver preparations. Immunoadsorption chromatography with PKlB bound to Sepharose isolated active enzyme which waa purified greater than 75-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of this pregaration in the presence of 2-mercaptoethanol demonstrated three polypeptides: M, 29,500; 32,500, and 34,000. Western blot analysis indicated that PKlB recognized an epitope which was found only on the M, 29,500 polypeptide. Two-dimensional gel electrophoresis 8ssaciated the enzymatic activity with this M, 29,600 band. High-pressure liquid chromatographic analysis of immmopurified enzyme defined three distinct, enzymatically active protein populations: I (M. 400,000 to 170,000); 11 (M. 130,000), and I11 (M, 43,000). An M,29,600 polypeptide was the sole constituent of Peaks I and III and a major constituent of Peak II. sedium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoeth-an01 indicated that in Peak 11, catalytically active M, 29,600 protein is associated with the other two polypeptides by diealffde bonds. In contrast, Peak I consists of a polymer of M, 29,500 polypeptide which is independent of disulfide interaction.
Intrahepatic enzymatic sulfation of bile acids has been proposed as an adaptive response to cholestasis (1). Sulfation increases the renal clearance of bile acids 200fold compared to the unsulfated species (2) and, thus, enables an alternative route of removal of the detergent bile acids from the hepatocyte in the presence of impaired bile flow. Present understanding of the control of this enzymatic pathway is rudimentary.