Kawaji A, Ohara K, Takabatake E
Department of Toxicology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Anal Biochem. 1993 Nov 1;214(2):409-12. doi: 10.1006/abio.1993.1515.
An assay system of flavin-containing monooxygenase was developed by fluorometric determination of benzydamine (BZY) N-oxidation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by rat liver microsomes was similar to that by purified FMO. The Km and Vmax values for the formation of N-desmethylbenzydamine (Nor-BZY) by rat liver microsomes were about 50 times greater and 2000 times less, respectively, than those of BZY N-oxide. Nor-BZY was not formed upon incubation with purified enzyme. BZY N-oxidation activity was completely inhibited both in the absence of NADPH and by heat inactivation. The reaction was inhibited in the presence of 0.5 mM thiourea, but 2 mM SKF-525A did not affect BZY N-oxidation. Moreover, rabbit antibody raised against the rat enzyme inhibited BZY N-oxidation. These results are in accord with a simple, rapid, and sensitive assay for the enzyme.
通过高效液相色谱荧光法测定苄达明（BZY）的N-氧化反应，建立了一种含黄素单加氧酶的检测系统。大鼠肝微粒体由BZY形成BZY N-氧化物的表观Km值与纯化的FMO的表观Km值相似。大鼠肝微粒体形成N-去甲基苄达明（Nor-BZY）的Km值和Vmax值分别比BZY N-氧化物的Km值和Vmax值大约大50倍和小2000倍。与纯化酶孵育时未形成Nor-BZY。在没有NADPH的情况下以及通过热失活，BZY N-氧化活性均被完全抑制。在0.5 mM硫脲存在下反应受到抑制，但2 mM SKF-525A不影响BZY N-氧化。此外，针对大鼠酶产生的兔抗体抑制BZY N-氧化。这些结果与该酶的一种简单、快速且灵敏的检测方法一致。
