Comparison of different activators and diazotization procedures in the assay of bilirubin-UDP-glucuronyl transferase activity in rat liver.
The activity of bilirubin-UDP-glucuronyl transferase (UDPGT) in rat liver microsomes was assayed in standardized incubation mixtures employing two different procedures to quantitate formation of conjugated bilirubin: solvent extraction and diazotization with sulfanilic acid, or selective coupling with diazotized ethyl anthranilate at pH 2.7. Both of these procedures yielded equivalent UDPGT activities with several different microsomal preparations, averaging, respectively, 385 and 372 microU protein with untreated microsomes, 646 and 608 microU protein following activation with UDP-N-acetylglucosamine (3.07 mmol/l), and 1370 and 1478 microU protein after treatment with digitonin (0.65%). In rats pretreated with phenobarbital (75 mg/kg per 6 days), a 70-90% increase in UDPGT activity was observed with either diazotization procedure, irrespective of whether 'native', UDP-N-acetylglucosamine-or digitonin-activated enzyme was employed. These studies indicate that, when other conditions are standardized, equivalent information about UDPGT can be obtained with either of two widely employed diazotization reactions and a variety of enzyme activation procedures.
D000818:Animals; D001557:Benzenesulfonates; D001663:Bilirubin / Q000031:analogs & derivatives / Q000378:metabolism; D003979:Diazonium Compounds; D004072:Digitonin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry; D013425:Sulfanilic Acids; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000494:pharmacology
D001557:Benzenesulfonates; D003979:Diazonium Compounds; D013425:Sulfanilic Acids; C017300:diazoethylanthranilate; C019270:bilirubin diglucuronide; D014537:Uridine Diphosphate N-Acetylglucosamine; D014453:Glucuronosyltransferase; D004072:Digitonin; D001663:Bilirubin; D010634:Phenobarbital; C000469:diazobenzenesulfonic acid