Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

Abstract

1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.

Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1

“…Bilirubin itself is highly water insoluble at pH values below 8 (Heirwegh et al, 1972). Thus, to achieve desired concentrations for in vitro assays, bilirubin is commonly dissolved in alkaline solutions, followed by pH adjustment 1908 ZHOU ET AL.…”

“…To address this issue, addition of albumin has been used to stabilize bilirubin solutions prepared in this manner (Black et al, 1970;Crawford et al, 1992). However, because bilirubin extensively binds to albumin, decreased rates of glucuronidation (Crawford et al, 1992) and non-Michaelis-Menten kinetics (Heirwegh et al, 1972) may be observed. In the present study, bilirubin was dissolved in 100% DMSO and stability was established for at least 8.5 h at room temperature and at least 7 days at Ϫ80°C.…”

NUCLEAR RECEPTOR, PREGNANE X RECEPTOR, IS REQUIRED FOR INDUCTION OF UDP-GLUCURONOSYLTRANSFERASES IN MOUSE LIVER BY PREGNENOLONE-16α-CARBONITRILE

“…The method of Heirwegh et al (1972) was followed to determine UGT activity toward bilirubin. The microsomal pellets were suspended in 0.25 M sucrose (0.4 g eq.…”

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

“…All enzyme assays were performed under linear conditions with respect to incubation time at 37°C and amount of protein. [15]; 0.35 mM bilirubin and 100 pM UDP-glucuronic acid (30 pCi/pmol) in 0.1 M triethanolamine/HCl, pH 7.4 [25]; 30 pM estriol and 50 pM UDP-glucuronic acid (30 pCi/ pmol) in 0.1 M Tris/HCl, pH 7.8 [24]. All other additions to assays were the same as described in the indicated published procedures except for L-a-phosphatidylcholine from egg yolk which was added to all assay mixtures as a freshly prepared dispersion in water to give a final concentration of 0.04% (massivol.).…”