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Rat hepatic bile acid sulfotransferase: Identification of the catalytic polypeptide and evidence for polymeric forms in female rats
Author(s) -
Collins Robert H.,
Lack Leon,
Harman Kenneth M.,
Killenberg Paul G.
Publication year - 1986
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840060406
Subject(s) - sodium dodecyl sulfate , gel electrophoresis , enzyme , biochemistry , chemistry , microbiology and biotechnology , polyacrylamide gel electrophoresis , sulfotransferase , sepharose , western blot , immunoadsorption , antibody , chromatography , biology , immunology , gene
Abstract 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: M r 29,500; 32,500, and 34,000. Western blot analysis indicated that PK1B recognized an epitope which was found only on the M r 29,500 polypeptide. Two‐dimensional gel electrophoresis associated the enzymatic activity with this M r 29,500 band. High‐pressure liquid chromatographic analysis of immunopurified enzyme defined three distinct, enzymatically active protein populations: I (M r 400,000 to 170,000); II (M r 130,000), and III (M r 43,000). An M r 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 M r 29,500 protein is associated with the other two polypeptides by disulfide bonds. In contrast, Peak I consists of a polymer of M r 29,500 polypeptide which is independent of disulfide interaction.