Investigation of the substrate specificity of a cloned expressed human bilirubin UDP-glucuronosyltransferase: UDP-sugar specificity and involvement in steroid and xenobiotic glucuronidation

S. B. Senafi, D. J. Clarke, B. Burchell

Research output: Contribution to journalArticle

223 Citations (Scopus)

Abstract

A cloned human bilirubin UDP-glucuronosyltransferase (UGT) stably expressed in Chinese hamster V79 cells was used to assess the substrate specificity of the enzyme. The catalytic potential (V(max.)/K(m(bilirubin))) of the enzyme with UDP-glucuronic acid (UDPGA) was 2-fold and 10-fold greater than that for UDP-xylose and UDP-glucose respectively. The formation of bilirubin mono- and di-conjugates was found to be dependent on time, UDP-sugar concentration and bilirubin concentration. Ex vivo studies demonstrated that the genetically engineered cell line was capable of the uptake and glucuronidation of bilirubin and the release of bilirubin glucuronide, indicating its usefulness in studying transport processes. Over 100 compounds, including drugs, xenobiotics and endogenous steroids, were tested as substrates for the enzyme to determine the chemical structures accepted as substrates. A wide diversity of xenobiotic compounds such as phenols, anthraquinones and flavones (many of which are in foodstuffs) were glucuronidated by the enzyme. The enzyme also had the capacity to glucuronidate oestriols and oestradiols sterioselectively. H.p.l.c. analysis of the regioselective glucuronidation of β-oestradiol (E2) demonstrated that it was conjugated solely at its A-ring hydroxy group by the bilirubin UGT to form E2-3-glucuronide, this was in contrast with human liver microsomes which formed 3- and 17-glucuronides of this oestrogen. Studies utilizing microsomes from a Crigler-Najjar patient and inhibition of E2 glucuronidation with bilirubin indicated that the cloned expressed bilirubin UGT was the major human UGT isoform responsible for the formation of E2-3-glucuronide, which is the predominant E2 conjugate in human urine.

Original languageEnglish
Pages (from-to)233-240
Number of pages8
JournalBiochemical Journal
Volume303
Issue number1
DOIs
Publication statusPublished - 1 Oct 1994
Externally publishedYes

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