IDENTIFICATION OF THE RABBIT LIVER UDP-GLUCURONOSYLTRANSFERASE CATALYZING THE GLUCURONIDATION OF 4-ETHOXYPHENYLUREA (DULCIN)
| Autor: | Kiminori Mohri, Adam G. Staines, Yoshihiro Uesawa, Brian Burchell, Audrey O'Sullivan |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Male
Gene isoform Spectrometry Mass Electrospray Ionization Magnetic Resonance Spectroscopy UGT1A4 Blotting Western Glucuronidation Pharmaceutical Science Dulcin In Vitro Techniques Transfection chemistry.chemical_compound Glucuronides Gallic Acid Chlorocebus aethiops Animals Enzyme Inhibitors Glucuronosyltransferase Chromatography High Pressure Liquid Pharmacology chemistry.chemical_classification Phenylurea Compounds Proteins Metabolism Glucuronic acid Isoenzymes Kinetics Enzyme Liver chemistry Biochemistry COS Cells Microsomes Liver Rabbits Glucuronide Chromatography Liquid |
| Zdroj: | Drug Metabolism and Disposition. 32:1476-1481 |
| ISSN: | 1521-009X 0090-9556 |
| DOI: | 10.1124/dmd.104.001206 |
| Popis: | Dulcin (DL), 4-ethoxyphenylurea, a synthetic chemical about 200 times as sweet as sucrose, has been proposed for use as an artificial sweetener. DL is excreted as a urinary ureido-N-glucuronide after oral administration to rabbits. The phenylurea N-glucuronide is the only ureido conjugate with glucuronic acid known at present; therefore, DL is interesting as a probe to search for new functions of UDP-glucuronosyltransferases (UGTs). Seven UGT isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT2B13, UGT2B14, and UGT2B16) have been identified from rabbit liver, but these UGTs have not been investigated using DL as a substrate. In this work, the identities of UGT isoforms catalyzing the formation of DL glucuronide were investigated using rabbit liver microsomes (RabLM) and cloned/expressed as rabbit UGT isoforms. DL-N-glucuronide (DNG) production was determined quantitatively in RabLM and homogenates of COS-7 cells expressing each UGT isoform by using electrospray liquid chromatography-tandem mass spectrometry. Analysis of DNG formation using RabLM, by Eadie-Hofstee plot, gave a Vmax of 0.911 nmol/min/mg protein and the Km of 1.66 mM. DNG formation was catalyzed only by cloned expressed rabbit UGT1A7 and UGT2B16 (Vmax of 3.98 and 1.16 pmol/min/mg protein and a Km of 1.23 and 1.69 mM, respectively). Substrate inhibition of UGT1A7 by octylgallate confirmed the significant contribution of UGT1A7 to the formation of DNG. Octylgallate was further shown to competitively inhibit DNG production by RabLM (Ki = 0.149 mM). These results demonstrate that UGT1A7 is the major isoform catalyzing the N-glucuronidation of DL in RabLM. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|