Molecular Properties of L-Kynurenine 3-Hydroxylase from Rat Liver Mitochondria
- 1 May 1977
- journal article
- research article
- Published by Oxford University Press (OUP) in The Journal of Biochemistry
- Vol. 81 (5), 1413-1425
- https://doi.org/10.1093/oxfordjournals.jbchem.a131595
Abstract
L-Kynurenine 3-hydroxylase [EC 1.14.1.2] has been solubilized with Triton X-100 and purified to homogeneity from rat liver mitochondria. The purified enzyme exists in aqueous media as an oligomeric aggregate and in 0.2% Triton X-100 as a dimer with a molecular weight of about 345,000. Its monomeric molecular weight is approximately 160,000, and 4 mol of FAD are associated with 1 mol of the monomeric unit of the enzyme. The addition of FAD to the apoenzyme resulted in remarkable increase in both L-kynurenine 3-hydroxylase activity and FAD fluorescence. The enzyme molecule migrated as a single band in polyacrylamide gel electrophoresis and in chromatography on a Sephadex G-200 column, and was resolved into two bands only after complete denaturation by high concentrations of SDS or urea. SDS-polyacrylamide gel electrophoresis in the presence of SDS at a concentration of more than 1% and Sephadex G-100 gel chromatography in the presence of 5 M urea caused cleavage of the monooxygenase molecule into a protein having a molecular weight of about 137,500 and a smaller fragment. Upon dissociation into these fragments, the enzyme activity was completely lost. The visible absorption spectrum of the oxidized holo-L-kynurenine 3-hydroxylase showed an absorption maximum at 408 nm with a prominent shoulder at around 445 nm. In contrast to the oxidized form, the dithionite-reduced form had peaks at about 420 and 554 nm. In spite of its electrophoretic homogeneity, appreciable amounts of cytochrome b5-like hemeprotein were detected in our highly purified preparation of L-kynurenine 3-hydroxylase. The purified L-kynurenine 3-hydroxylase catalyzed the reductions by NADH or NADPH of DCPI and ferricyanide.This publication has 6 references indexed in Scilit:
- Possible Involvement of Cytochrome b5in L-Kynurenine-3-Hydroxylase of Rat Liver MitochondriaHoppe-Seyler´s Zeitschrift Für Physiologische Chemie, 1968
- ZUR LIPOIDABHANGIGKEIT DER L-KYNURENIN-3-HYDROXYLASE1967
- The Reversible Conversion of Lipoyl Dehydrogenase to an Artifactual Enzyme by Oxidation of Sulfhydryl GroupsJournal of Biological Chemistry, 1966
- The gel-filtration behaviour of proteins related to their molecular weights over a wide rangeBiochemical Journal, 1965
- THE ROLE OF RIBOFLAVIN AND VITAMIN B6 IN TRYPTOPHAN METABOLISMJournal of Biological Chemistry, 1955
- PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENTJournal of Biological Chemistry, 1951