Kynurenine 3-monoxygenase is present in the outer membrane of rat liver mitochondria, where it catalyzes the oxidation of L-kynurenine to 3-hydroxy-L-kynurenine. This assay follows the disappearance of the substrate, l-[G-3H]kynurenine.
Kynurenine was separated from L-3-hydroxykynurenine by chromatography on a Waters /uJBondapak Clg column (8 mm X 100 mm, 10-pi.m), using a flow rate of 3 mL/min. The mobile phase was 0.02 M sodium acetate (pH 5.5) containing 2% methanol. Radioactivity was quantitated with a flow-through detector, using scintillation fluid at 3 mL/min.
The reaction mixture contained 0.1 M Tris acetate (pH 8.0), 10 mM KC1, 1 mM EDTA, 3 mM glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehy-
drogenase, 50 to 250 ju,M L-[G-3H]kynurenine (0.04 /¿Ci/mL), 100 ¡xM NADPH, and enzyme (0.25-0.5 mg/mL). After 30 minutes of incubation at 37°C, assays were quenched with the addition of 5 mL of acetic acid per milliliter and heating for 2 minutes at 95°C. After centrifugation, a 0.4 mL aliquot was analyzed by chromatography. The assay was linear with time up to 120 minutes and with protein concentration up to 1 mg/mL.
Rat liver mitochondria were isolated in 0.25 M sucrose by standard procedures.
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