Assay of tryptophan 2,3-dioxygenase using liver slices and high-performance liquid chromatography.
Liver tryptophan 2,3-dioxygenase (TDO) activity was determined by high-performance liquid chromatography. The enzyme activity was expressed as the sum of N-formyl-L-kynurenine (FK) and L-kynurenine (KYN) produced from L-tryptophan (TRY) by liver slices. FK and KYN were detected spectrophotometrically at 254 nm after their separation on a reversed-phase C18 column. KYN formation proceeded according to zero-order kinetics for at least 4 h with 15 mM TRY at 37 degrees C. The apparent Michaelis constant was 1.2 mM TRY with a maximum velocity of 59 pmol min-1 mg-1 wet weight. The method was applied for TDO assay in mice treated with the organophosphorus acid triester diazinon. Kynurenine formamidase inhibition by diazinon resulted in reduced KYN formation, FK accumulation, and moderate TDO increase.
Seifert
J
J
Department of Environmental Biochemistry, University of Hawaii, Honolulu 96822.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D007737:Kynurenine / Q000031:analogs & derivatives / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D013053:Spectrophotometry; D014364:Tryptophan / Q000378:metabolism; D014366:Tryptophan Oxygenase / Q000032:analysis* / Q000378:metabolism
C007772:N'-formylkynurenine; D007737:Kynurenine; D014364:Tryptophan; D014366:Tryptophan Oxygenase