使用肝切片和高效液相色谱法测定色氨酸2,3-双加氧酶

Assay of tryptophan 2,3-dioxygenase using liver slices and high-performance liquid chromatography.

作者信息

Seifert J

机构信息

Department of Environmental Biochemistry, University of Hawaii, Honolulu 96822.

出版信息

J Chromatogr. 1993 May 5;614(2):227-31. doi: 10.1016/0378-4347(93)80313-s.

DOI:10.1016/0378-4347(93)80313-s

PMID:8314934

Abstract

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.

摘要

通过高效液相色谱法测定肝脏色氨酸2,3-双加氧酶（TDO）的活性。酶活性以肝脏切片将L-色氨酸（TRY）转化产生的N-甲酰基-L-犬尿氨酸（FK）和L-犬尿氨酸（KYN）的总量表示。FK和KYN在反相C18柱上分离后，于254 nm处通过分光光度法进行检测。在37℃下，15 mM TRY存在时，KYN的生成至少4小时呈零级动力学。表观米氏常数为1.2 mM TRY，最大速度为59 pmol min-1 mg-1湿重。该方法应用于用有机磷酸三酯二嗪农处理的小鼠的TDO测定。二嗪农对犬尿氨酸甲酰胺酶的抑制导致KYN生成减少、FK积累以及TDO适度增加。