Wu R F, Ichikawa Y
Department of Biochemistry, Kagawa Medical School, Japan.
Biochim Biophys Acta. 1994 Oct 19;1208(2):204-10. doi: 10.1016/0167-4838(94)90105-8.
An FAD-containing monooxygenase (EC 1.14.13.8) was purified from porcine liver microsomes by a new purification procedure and confirmed to give an electrophoretically single protein band. The optical and CD spectra, fluorescence and molar extinction coefficients of the FMO were investigated. The activity of the FMO was examined kinetically with neurotoxins, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ), and 1-methyl-6,7-dihydroxytetrahydroisoquinoline (MDTIQ), as substrates. The kinetic parameters of the FMO for the neurotoxins, molecular oxygen and electron donors were determined, in comparison with those of dimethylaniline. The CD spectrum of the FMO was measured in the absence and presence of NADP+, dimethylaniline or both. The results showed that the FMO metabolized the neurotoxins, and that NADH was a weak electron donor for it. The CD spectrum of the FMO in the oxidized form, which acts as an oxidase and oxygenase, unlike that of D-amino-acid oxidase, showed negative ellipticity, the secondary structure of the FMO changed, and the alpha-helix structure of the monooxygenase was affected by the formation of a complex of the FMO with NADP+, DMA or both.
通过一种新的纯化方法从猪肝微粒体中纯化出一种含黄素腺嘌呤二核苷酸（FAD）的单加氧酶（EC 1.14.13.8），并确认其在电泳中呈现单一蛋白条带。对该FMO的光学光谱和圆二色光谱、荧光以及摩尔消光系数进行了研究。以神经毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）、1,2,3,4-四氢异喹啉（TIQ）和1-甲基-6,7-二羟基四氢异喹啉（MDTIQ）作为底物，对FMO的活性进行了动力学检测。与二甲基苯胺相比，测定了FMO对神经毒素、分子氧和电子供体的动力学参数。在不存在和存在NADP⁺、二甲基苯胺或两者的情况下测量了FMO的圆二色光谱。结果表明，FMO可代谢神经毒素，且NADH是其较弱的电子供体。与D-氨基酸氧化酶不同，作为氧化酶和加氧酶的氧化形式的FMO的圆二色光谱显示出负椭圆率，FMO的二级结构发生了变化，并且FMO与NADP⁺、DMA或两者形成复合物会影响单加氧酶的α-螺旋结构。
