Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes

Abstract

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.

Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase

“…Further, the initial rates of formation of the two isomeric (C-8, C-12) BMGs by these microsomal preparations indicated that the C-8 isomer was produced approximately twice as rapidly as the C-12 isomer (Table 1), confirming previous work of Blanckaert et al (1979). Gunn-rat liver microsomes, used to reconstitute purified transferase preparations (Burchell, 1982), were also examined for their ability to catalyse the formation of bilirubin glucuronides. Gunn-rat liver microsomes alone, incubated in the presence or absence of 0.1% (w/v) Lubrol, were unable to catalyse the formation of BMG and the conversion of BMG into BDG, confirming the results obtained by Blanckaert et al (1979) and Sieg et al (1982).…”

“…Bilirubin UDP-glucuronyltransferase activity was 40-S50nmol of total bilirubin glucuronides formed/min per mg of protein when the purified enzyme was assayed in the presence of Gunn-rat liver microsomes by the diazo-method or the radioassay. The final specific activity of the purified enzyme could be higher (Burchell, 1982), Fig. 1.…”

“…Bilirubin UDP-glucuronyltransferase was then purified 55-fold [over the (NH4)2SO4 fraction of Wistar-rat liver microsomes previously described by Burchell (1982)], and the results are summarized in Table 1.…”

“…Microsomal and purified bilirubin UDP-glucuronyltransferase were prepared from liver of male Wistar rats pretreated with 2g of phenobarbital/ litre in the drinking water for 7 days, by methods previously described (Burchell, 1977(Burchell, , 1980(Burchell, , 1981b. Purified enzyme fractions were reconstituted with Gunn-rat liver microsomes before assay (Burchell, 1982).…”

Molecular basis of bilirubin UDP-glucuronosyltransferase induction in spontaneously diabetic rats, acetone-treated rats and starved rats

“…Anti-UGT1A1 antibody (R "! ) was raised in White Lopeared rabbits by the method described by Burchell [14] by using highly purified rat liver UGT1A1 [15].…”