A rapid and sensitive method for the assay of UDP-glucuronosyltransferase activity toward bile acids.
A rapid and sensitive procedure is described for the assay of rat liver microsomal UDP-glucuronosyltransferase activity toward the bile acids chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid using the radioactively labeled bile acids as substrates. The unreacted bile acids were separated from the bile acid glucuronides formed as products of the enzymatic reactions by extraction with chloroform, leaving the bile acid glucuronides in the aqueous phases. The bile acid glucuronides were characterized by their mobilities in thin-layer chromatography and identified by their sensitivity to hydrolysis with beta-glucuronidase and inhibition of hydrolysis by the specific beta-glucuronidase inhibitor D-saccharic acid-1,4-lactone. Enzyme activities were optimal at pH 6.8 and were maximally stimulated about fourfold by the addition of the nonionic detergent Brij 58 at a concentration of 0.3 mg/mg microsomal protein. The kinetic parameters for the various bile acids as substrates were determined.
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D005965:Glucuronates / Q000096:biosynthesis; D014453:Glucuronosyltransferase / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D005965:Glucuronates; D014453:Glucuronosyltransferase