Isolation and characterization of hyodeoxycholic-acid: UDP-glucuronosyltransferase from human liver

Eur J Biochem. 1991 Sep 1;200(2):393-400. doi: 10.1111/j.1432-1033.1991.tb16197.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bile Acids and Salts / metabolism
  • Chromatography, Liquid
  • Deoxycholic Acid / isolation & purification*
  • Deoxycholic Acid / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Glucuronosyltransferase / isolation & purification*
  • Glucuronosyltransferase / metabolism
  • Humans
  • Kinetics
  • Microsomes, Liver / enzymology*
  • Phospholipids / metabolism
  • Substrate Specificity
  • Testosterone / metabolism

Substances

  • Bile Acids and Salts
  • Phospholipids
  • Deoxycholic Acid
  • Testosterone
  • hyodeoxycholic acid
  • Glucuronosyltransferase