Isolation and characterization of hyodeoxycholic-acid: UDP-glucuronosyltransferase from human liver.
The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.
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Department of Internal Medicine III, Aachen University of Technology, Federal Republic of Germany.
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D001647:Bile Acids and Salts / Q000378:metabolism; D002853:Chromatography, Liquid; D003840:Deoxycholic Acid / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D001647:Bile Acids and Salts; D010743:Phospholipids; D003840:Deoxycholic Acid; D013739:Testosterone; C010471:hyodeoxycholic acid; D014453:Glucuronosyltransferase