HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708.
The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.
D000907:Antibodies, Bacterial / Q000302:isolation & purification*; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005051:Eubacterium / Q000276:immunology* / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases; D005779:Immunodiffusion; D007074:Immunoglobulin G / Q000302:isolation & purification; D008970:Molecular Weight; D010088:Oxidoreductases; D010455:Peptides / Q000276:immunology*; D011233:Precipitin Tests; D013250:Steroid Hydroxylases / Q000032:analysis
D000907:Antibodies, Bacterial; D002793:Cholic Acids; D007074:Immunoglobulin G; D010455:Peptides; D010088:Oxidoreductases; D006913:Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase; D013250:Steroid Hydroxylases; D019826:Cholic Acid