A rapid NAD+-linked assay for microsomal uridine diphosphate glucuronyltransferase of rat liver and some observations on substrate specificity of the enzyme.
1. A new and rapid continuous assay of rat liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17) has been developed. It is based on measurement of UDP production from UDP-glucuronate during the glucuronidation reaction; UDP production was continuously measured by coupling it to the conversion of NADH into NAD+ through pyruvate kinase and lactate dehydrogenase. This assay is independent of the acceptor substrate used; several findings confirm its applicability. 2. The glucuronidation rate of a series of phenol derivatives was determined with this assay, by using a Triton X-100-activated microsomal preparation as enzyme source. Conjugation of a series of nitrophenol derivatives was also investigated by the 'classical' assay (measurement of disappearance of the yellow colour of the nitrophenol during glucuronidation). The substrate with the highest conversion rate was 3-methyl-2-nitrophenol. 3. Both electron releasing and electron withdrawing ring substituents increased the glucuronidation rate of the phenol derivatives, as compared with phenol. 4. Lipid solubility seems important for determining the conversion rate: poorly lipid-soluble substrates were glucuronidated only at a low rate and high lipid solubility seems to be a prerequisite for high conversion rate. Glucuronidation of poorly lipid-soluble compounds may be limited by diffusion. 5. The consequences of these findings for the interpretation of studies on heterogeneity of the enzyme are discussed.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D005966:Glucuronidase; D014453:Glucuronosyltransferase / Q000032:analysis*; D006602:Hexosyltransferases / Q000032:analysis*; D007700:Kinetics; D008055:Lipids; D008862:Microsomes, Liver / Q000032:analysis / Q000201:enzymology*; D009243:NAD / Q000032:analysis; D009596:Nitrophenols; D051381:Rats; D012995:Solubility
D008055:Lipids; D009596:Nitrophenols; D009243:NAD; D006602:Hexosyltransferases; D014453:Glucuronosyltransferase; D005966:Glucuronidase