Details

Autor(en) / Beteiligte

• Matern, H.
• Heinemann, H.
• Matern, S.
• Bongartz, M.

Titel

Radioassay of UDP-Glucuronosyltransferase Activities toward Endogenous Substrates Using Labeled UDP-Glucuronic Acid and an Organic Solvent Extraction Procedure

Ist Teil von

• Analytical biochemistry, 1994-06, Vol.219 (2), p.182-188

Ort / Verlag

San Diego, CA: Elsevier Inc

Erscheinungsjahr

1994

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• A rapid and sensitive radioassay for measuring UDP-glucuronosyltransferase activities (EC 2.4.1.17) toward the major endogenous substrates hyodeoxycholic and hyocholic acids, bilirubin, estriol, androsterone, and testosterone has been developed. In this assay, 14C-labeled glucuronides are formed from the enzyme-catalyzed reaction of 14C-labeled UDP-glucuronic acid with the unlabeled aglycones. Following incubation, the 14C-labeled glucuronides are separated under acidic conditions from the unreacted 14C-labeled UDP-glucuronic acid by a single extraction with ethyl acetate. The recovery of glucuronides into ethyl acetate was greater than 90%, whereas the carryover of unreacted UDP-glucuronic acid into the organic phase was approximately 0.2%. The reaction products extracted into ethyl acetate were characterized by their mobilities in thin-layer chromatography and identified as glucuronides by their sensitivity to hydrolysis with β-glucuronidase and inhibition of hydrolysis by the specific β-glucuronidase inhibitor D-saccharic acid-1,4-lactone. The optimal conditions of enzyme reactions with the individual aglycones have been defined with human liver microsomes as enzyme source. For all aglycones investigated, 10-30 μg of microsomal protein are sufficient for enzyme estimation. The assay is applicable to biochemical studies of UDP-glucuronosyltransferases, as well as to measurement of these enzyme activities from small amounts of clinical liver specimens.