A radiometric assay for kynurenine 3-hydroxylase based on the release of 3H2O during hydroxylation of L-[3,5-3H]kynurenine.

Published

Journal Article

A rapid and sensitive assay for kynurenine 3-hydroxylase (KH) has been developed. This radiometric assay is based on the enzymatic synthesis of tritiated water from L-[3,5-3H]kynurenine during the hydroxylation reaction. Radiolabeled water is quantified following selective adsorption of the isotopic substrate and its metabolite with activated charcoal. The assay is suitable for detecting 0.1 pmol enzyme activity per minute per milligram protein in tissues displaying low levels of the enzyme. The amount of water produced in the reaction, as calculated from the tritium released, was stoichiometric with the 3-hydroxykynurenine product detected by HPLC. Rat liver KH was characterized by cofactor specificity and kinetic parameters. NADPH was preferred over NADH as coreductant in the reaction. Tetrahydrobiopterin was not a cofactor. The tissue distribution of KH activity in the rat suggested that the majority of active enzyme is located in liver and kidney. Detectable amounts were found in several other tissues, including brain which had low but significant levels of activity in every region assayed.

Full Text

Cited Authors

  • Erickson, JB; Flanagan, EM; Russo, S; Reinhard, JF

Published Date

  • September 1992

Published In

Volume / Issue

  • 205 / 2

Start / End Page

  • 257 - 262

PubMed ID

  • 1332540

Pubmed Central ID

  • 1332540

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1016/0003-2697(92)90432-7

Language

  • eng

Conference Location

  • United States