This chapter presents the assay, purification, and properties of kynurenine 3-monooxygenase. It is localized in the outer membrane fraction of rat liver mitochondria. A 12-fold concentration of the enzyme activity as compared with that of the original mitochondrial preparations has been obtained by digitonin treatment and differential centrifugation. The enzyme activity can be determined spectrophotometrically by measuring the disappearance of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 mμ in the presence of L-kynurenine. All purification procedures are generally carried out at 0–4º. Intact mitochondria catalyze the kynurenine-dependent NADPH oxidation approximately as rapidly as hypotonically treated mitochondria. This result is consistent with the localization of kynurenine hydroxylase in the outer membrane of mitochondria. When the 105,000 g pellet is treated with ammonium sulfate at pH 4.5, the treated fraction by itself has 60% of the original activity. The full activity is almost completely recovered upon the addition of 10 μM flavin adenine dinucleotide (FAD).
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