Bilirubin glucuronyltransferase. Specific assay and kinetic studies.
1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg(2+) and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 2.4.1.17). 3. This diazo compound was labelled when [U-(14)C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with beta-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ;equivalents' conjugated/h per mg of protein and 16.9-18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The K(m) values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3x10(-4)m and 1.67x10(-3)m, respectively. 7. The addition of Mg(2+) at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1-20mm has no effect on the glucuronidation of bilirubin in vitro.
D000818:Animals; D001391:Azo Compounds; D001663:Bilirubin / Q000096:biosynthesis / Q000378:metabolism; D002247:Carbon Isotopes; D002854:Chromatography, Paper; D002855:Chromatography, Thin Layer; D005965:Glucuronates / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D006602:Hexosyltransferases / Q000032:analysis* / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium; D008862:Microsomes, Liver / Q000201:enzymology; D009602:Nitrosamines / Q000494:pharmacology; D051381:Rats; D012908:Snails; D014500:Uracil Nucleotides
D001391:Azo Compounds; D002247:Carbon Isotopes; D005965:Glucuronates; D009602:Nitrosamines; D014500:Uracil Nucleotides; D006602:Hexosyltransferases; D005966:Glucuronidase; D008274:Magnesium; D001663:Bilirubin