L-kynurenine 3-monooxygenase from mitochondrial outer membrane of pig liver: purification, some properties, and monoclonal antibodies directed to the enzyme.
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 beta-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 sulfate-polyacrylamide 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.
Uemura
T
T
Department of Neurochemistry, Tokyo Institute of Psychiatry. uemura@prit.go.jp
Hirai
K
K
D000645:Ammonium Sulfate; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000494:pharmacology*; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005960:Glucosides; D015151:Immunoblotting; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology; D050601:Kynurenine 3-Monooxygenase; D008930:Mitochondria, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D011817:Rabbits; D012685:Sepharose / Q000031:analogs & derivatives; D013053:Spectrophotometry; D013552:Swine
D000911:Antibodies, Monoclonal; D005960:Glucosides; D007202:Indicators and Reagents; D005182:Flavin-Adenine Dinucleotide; C018619:octyl-beta-D-glucoside; C043982:DEAE-Sepharose CL-6B; D012685:Sepharose; D006899:Mixed Function Oxygenases; D050601:Kynurenine 3-Monooxygenase; D000645:Ammonium Sulfate