Erickson J B, Flanagan E M, Russo S, Reinhard J F
Division of Pharmacology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Anal Biochem. 1992 Sep;205(2):257-62. doi: 10.1016/0003-2697(92)90432-7.
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.
已开发出一种用于犬尿氨酸3-羟化酶（KH）的快速灵敏检测方法。这种放射性检测方法基于在羟基化反应过程中由L-[3,5-³H]犬尿氨酸酶促合成氚化水。在用活性炭选择性吸附同位素底物及其代谢物后，对放射性标记的水进行定量。该检测方法适用于检测每毫克蛋白质每分钟0.1皮摩尔酶活性的低水平表达该酶的组织。根据释放的氚计算出的反应中产生的水量与通过高效液相色谱法检测到的3-羟基犬尿氨酸产物呈化学计量关系。通过辅因子特异性和动力学参数对大鼠肝脏KH进行了表征。在该反应中，NADPH比NADH更适合作为辅酶。四氢生物蝶呤不是辅因子。大鼠体内KH活性的组织分布表明，大部分活性酶位于肝脏和肾脏。在其他几个组织中也检测到了可检测量，包括大脑，在每个检测区域其活性水平较低但很显著。
