L-Kynurenine 3-Monooxygenase from Mitochondrial Outer Membrane of Pig Liver: Purification, Some Properties, and Monoclonal Antibodies Directed to the Enzyme.
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- Uemura Tomihiko
- Department of Neurochemistry, Tokyo Institute of Psychiatry
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- Hirai Kumiko
- Department of Neurochemistry, Tokyo Institute of Psychiatry
Bibliographic Information
- Other Title
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- L Kynurenine 3 Monooxygenase from Mitoc
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Abstract
We have purified L-kynurenine 3-monooxygenase from pig liver mitochondria using a procedure involving seven steps composed of (1) preparation of mitochondrial outer membrane, (2) preparation of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio] -1-propane sulfonate (Chaps)-insoluble outer membrane material, (3) extraction of the enzyme with β-octylglucoside, (4) ammonium sulfate fractionation, (5) DEAE-Sepharose CL-6B chromatography, (6) Matrex gel orange A affinity chromatography, and (7) high-performance liquid chromatography (HPLC) gel filtration. The final preparation had an about 160-fold purified enzyme activity with a yield of 0.8%. The apparent molecular mass of the aggregated form of the native enzyme was determined to be close to 300 kDa by HPLC gel filtration in the presence of 0.005% Triton X-100. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) showed a main protein band with an apparent molecular mass of about 49 kDa. The enzyme was found to be about 86% pure by the criterion of SDS-PAGE. The dissociated form of the enzyme contains 1 mol of non-covalently bound FAD/mol of protein monomer. The UV/visible spectrum had absorption peaks at 275, 384, and 450 nm, typical of a simple flavoprotein. Five inhibitory monoclonal antibodies against the enzyme were obtained. They could stain moderately a single protein band (49 kDa) in a Western blot.
Journal
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- The Journal of Biochemistry
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The Journal of Biochemistry 123 (2), 253-262, 1998
The Japanese Biochemical Society
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Details 詳細情報について
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- CRID
- 1390001204929990784
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- NII Article ID
- 130003533356
- 10005841593
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- NII Book ID
- AA00694073
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- COI
- 1:CAS:528:DyaK1cXit1GlsLg%3D
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- ISSN
- 17562651
- 0021924X
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- NDL BIB ID
- 4406086
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- PubMed
- 9538200
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- PubMed
- CiNii Articles
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- Abstract License Flag
- Disallowed