Kynureninase [L-kynurenine hydrolase EC 3.7.1.3] has been purified 614-fold from rat liver cytosol. The purification procedure involved pH treatment, ammonium sulfate fractionation, Sephadex G-100 gel filtration, DEAE-Sepharose CL-6B chromatography, extraction with 40% saturated solution of ammonium sulfate and hydroxyapatite chromatography. The enzyme was found to be homogeneous by the criteria of disc gel electrophoresis. SDS-gel electrophoresis and sucrose gradient centrifugation. The enzyme was obtained as a holo-enzyme which showed an absorption maximum at 420 nm. In the absence of pyridoxal 5-phosphate (PLP) the enzyme was dissociated into an apoenzyme. The isoelectric points of holoenzyme and apoenzyme are 5.7 and 6.1. Sucrose gradient centrifugation and SDS-gel electrophoresis gave molecular weight estimates of 95,000 and 55,000, respectively. The optimum pH shifted to higher pH with increase in the concentration of PLP. The Michaelis constants were determined as follows: kynurenine, 240 microM; 3-hydroxykynurenine, 13 microM; PLP, 0.1-1.7 microM. The maximum velocity for 3-hydroxykynurenine was 11 times higher than that for kynurenine at pH 7.7. This enzyme can be regarded as a 3-hydroxykynureninase type enzyme.

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Tryptophan contents of liver, serum and kidney were determined in normal and vitamin-B-6-deficient rats after tryptophan injection. Tryptophan contents of normal and B-6-deficient liver were different, but not those in serum and kidney. Both kynurenine and 3-hydroxykynurenine accumulated in B-6-deficient liver more than in the normal. The 3-hydroxykynurenine contents after tryptophan injection (30 mg/100 g body wt.) increased to 1380 nmol/g of liver at 1-1.5 h, a value sufficient to produce xanthurenate, in view of the Km value of kynurenine aminotransferase. The enzymes metabolizing kynurenine were assayed at various times after tryptophan injection. The activity of kynureninase holoenzyme in B-6-deficient liver was much decreased, but the activity of total enzyme was not changed. It appeared that a high dose of tryptophan in B-6-deficient rats could cause a greater deficiency of pyridoxal 5-phosphate. Tryptophan metabolism in B-6-deficient rat liver after tryptophan administration is discussed.

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