Pharmacokinetics, Dynamics and Metabolism, Chesterfield, MO 63017, USA.
Drug Metab Dispos. 2009 Oct;37(10):1987-90. doi: 10.1124/dmd.109.027714. Epub 2009 Jul 27.
Mammalian flavin-containing monooxygenase (FMO) enzymes catalyze oxidation at nucleophilic, heteroatom centers and are important for drug, xenobiotic, and endogenous substrate metabolism. In human liver, human FMO3 (hFMO3) is the most abundant FMO isoform and is known to contribute to the hepatic clearance of a variety of clinical drugs. The purpose of the current study was to express and compare the dog (beagle) FMO3 (dFMO3) to hFMO3. A full-length dFMO3 cDNA was obtained from liver by reverse transcription-polymerase chain reaction. Using a baculovirus expression system in Spodoptera frugiperda insect cells, dFMO3 was expressed to protein levels of 0.50 nmol/mg, as determined by liquid chromatography-fluorescence detection. Expressed dFMO3 displayed Michaelis-Menten kinetics, catalyzing NADPH-dependent N-oxidation of benzydamine, with K(m) and V(max) values of 18.6 microM and 0.63 nmol N-oxide formed/min/nmol of enzyme, respectively. Benzydamine N-oxidation catalyzed by hFMO3 showed values of 42.6 microM (K(m)) and 3.56 nmol/min/nmol of enzyme (V(max)). Human FMO3 was observed to catalyze the S-oxidation of sulindac sulfide, with respective K(m) and V(max) values of 69.3 microM and 35.4 nmol/min/nmol of enzyme. dFMO3 also catalyzed sulindac sulfide S-oxidation with 6.8 nmol/min/nmol of enzyme being the highest velocity observed. Finally, Western blot analysis indicated protein expression levels of dFMO3 in pooled dog liver and lung microsomes to be 27 and 9 pmol/mg, respectively. In summary, dFMO3 appears to be a functional enzyme expressed at appreciable levels in liver, but one with some kinetic properties that are substantially different from its human homolog hFMO3.
哺乳动物黄素单加氧酶 (FMO) 酶催化亲核、杂原子中心的氧化，对于药物、外源性物质和内源性底物代谢非常重要。在人肝中，人黄素单加氧酶 3 (hFMO3) 是最丰富的 FMO 同工酶，已知其有助于多种临床药物的肝清除。本研究的目的是表达和比较犬（比格犬）黄素单加氧酶 3 (dFMO3) 与人黄素单加氧酶 3 (hFMO3)。通过反转录-聚合酶链反应从肝中获得全长 dFMO3 cDNA。使用杆状病毒表达系统在 Spodoptera frugiperda 昆虫细胞中表达 dFMO3，表达水平为 0.50 nmol/mg，通过液相色谱-荧光检测确定。表达的 dFMO3 显示出 Michaelis-Menten 动力学，催化苯并达明的 NADPH 依赖性 N-氧化，K(m) 和 V(max) 值分别为 18.6 μM 和 0.63 nmol N-氧化物形成/min/nmol 酶。hFMO3 催化的苯并达明 N-氧化显示出 42.6 μM (K(m)) 和 3.56 nmol/min/nmol 酶 (V(max))。观察到 hFMO3 催化舒林酸硫醚的 S-氧化，相应的 K(m) 和 V(max) 值分别为 69.3 μM 和 35.4 nmol/min/nmol 酶。dFMO3 也催化舒林酸硫醚的 S-氧化，观察到的最高速度为 6.8 nmol/min/nmol 酶。最后，Western blot 分析表明，在狗肝和肺微粒体的混合样本中，dFMO3 的蛋白表达水平分别为 27 和 9 pmol/mg。总之，dFMO3 似乎是一种功能性酶，在肝中以相当高的水平表达，但与人类同源物 hFMO3 的动力学特性有很大不同。
