Rat hepatic bile acid sulfotransferase: identification of the catalytic polypeptide and evidence for polymeric forms in female rats.
A monoclonal antibody, PK1B, directed against rat liver bile acid sulfotransferase was used for the purification and characterization of the enzyme. Incubation of rat liver supernatant with the antibody followed by immunoprecipitation with Staphylococcus aureus cells demonstrated that PK1B 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 PK1B bound to Sepharose isolated active enzyme which was purified greater than 75-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of this preparation in the presence of 2-mercaptoethanol demonstrated three polypeptides: Mr 29,500; 32,500, and 34,000. Western blot analysis indicated that PK1B recognized an epitope which was found only on the Mr 29,500 polypeptide. Two-dimensional gel electrophoresis associated the enzymatic activity with this Mr 29,500 band. High-pressure liquid chromatographic analysis of immunopurified enzyme defined three distinct, enzymatically active protein populations: I (Mr 400,000 to 170,000); II (Mr 130,000), and III (Mr 43,000). An Mr 29,500 polypeptide was the sole constituent of Peaks I and III and a major constituent of Peak II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol indicated that in Peak II, catalytically active Mr 29,500 protein is associated with the other two polypeptides by disulfide bonds. In contrast, Peak I consists of a polymer of Mr 29,500 polypeptide which is independent of disulfide interaction.
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007163:Immunosorbent Techniques; D008099:Liver / Q000201:enzymology*; D010455:Peptides / Q000032:analysis; D011108:Polymers / Q000032:analysis; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D012685:Sepharose / Q000031:analogs & derivatives; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D010455:Peptides; D011108:Polymers; C017842:cibacron blue-sepharose; D012685:Sepharose; D013466:Sulfurtransferases; D015238:Sulfotransferases; C019786:bile-salt sulfotransferase