Expression and characterization of functional dog flavin-containing monooxygenase 1.
Expression and characterization of functional dog flavin-containing monooxygenase 1.
复制标题
功能性狗含黄素单加氧酶的表达和表征1。
作者：
J. Stevens;R. Melton;M. J. Zaya;L. Engel
用逆转录-聚合酶链反应（RT-PCR）方法从犬肝脏中获得了含黄素单加氧酶1（FMO 1）的全长cDNA（dFMO 1）。dFMO 1的氨基酸序列与人FMO 1的同源性为89%。在Sf-9昆虫细胞中使用杆状病毒表达系统，dFMO 1表达至0.4 nmol/mg的蛋白水平，如通过免疫定量测定的。表达的酶的黄素含量与免疫检测的dFMO 1蛋白水平一致。表达的dFMO 1催化NADPH依赖性甲基对甲苯基硫醚氧化，K（m）和V（max）值分别为98.6 μ M和63.8 nmol S-氧化物形成/min/mg蛋白质。相比之下，人FMO 1显示出相似的值87.1 μ M（K（m））和51.0 nmol/min/mg（V（max））。dFMO 1的活性表现出特征性的pH依赖性，随着孵育pH从7.6增加到9.0，S-氧化酶活性增加4.5倍。人FMO 1也显示出反应速率随pH的增加，但最适值略低，为8.0至8.4。dFMO 1还催化丙咪嗪N-氧化，K（m）为4.7 μ M，V（max）为82.1 nmol/min/mg蛋白质。该酶显示出FMO酶的其他特征，在不存在NADPH的情况下加热时酶活性迅速耗尽。合并的肝微粒体样本中，蛋白水平为74 pmol dFMO 1/mg微粒体蛋白，表明该酶是一种主要的犬肝单加氧酶。总之，催化活性dFMO 1的表达和表征将允许确定该酶在异生物质代谢中的作用。
A full-length dog (beagle) flavin-containing monooxygenase 1 (FMO1) cDNA (dFMO1) was obtained from liver by reverse transcription-polymerase chain reaction. The amino acid sequence of dFMO1 was 89% homologous to human FMO1. Using a baculovirus expression system in Sf-9 insect cells, dFMO1 was expressed to protein levels of 0.4 nmol/mg, as determined by immunoquantitation. The flavin content of the expressed enzyme was consistent with immunodetectable dFMO1 protein levels. Expressed dFMO1 catalyzed NADPH-dependent methyl p-tolyl sulfide oxidation, with K(m) and V(max) values of 98.6 microM and 63.8 nmol of S-oxide formed/min/mg of protein, respectively. By comparison, human FMO1 showed similar values of 87.1 microM (K(m)) and 51.0 nmol/min/mg (V(max)). Activity for dFMO1 showed characteristic pH dependence, with a 4.5-fold increase in S-oxidase activity as the incubation pH increased from 7.6 to 9.0. Human FMO1 also showed an increase in reaction rate with pH but a somewhat lower optimum of 8.0 to 8.4. dFMO1 also catalyzed imipramine N-oxidation, with a K(m) of 4.7 microM and a V(max) of 82.1 nmol/min/mg of protein. This enzyme displayed other characteristics of FMO enzymes, with rapid depletion of enzyme activity upon heating in the absence of NADPH. Protein levels of 74 pmol of dFMO1/mg of microsomal protein were determined for a pooled liver microsome sample, suggesting that this enzyme is a major canine hepatic monooxygenase. In conclusion, the expression and characterization of catalytically active dFMO1 will allow the role of this enzyme in the metabolism of xenobiotics to be determined.
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影响因子：
4.1
作者：
CASHMAN, JR;PARK, SB;BENOWITZ, NL
通讯作者：
BENOWITZ, NL
人含黄素单加氧酶：底物特异性及其在药物代谢中的作用。
影响因子：
2.3
作者：
Cashman,JR
通讯作者：
Cashman,JR
影响因子：
5.8
作者：
Lang, DH;Yeung, CK;Rettie, AE
通讯作者：
Rettie, AE
人含黄素单加氧酶 (FMO) 潜在体内探针的体外评估：FMO 和 P450 亚型对苯甲胺和咖啡因的代谢。
影响因子：
3.4
作者：
Lang,DH;Rettie,AE
通讯作者：
Rettie,AE
人含黄素单加氧酶的立体选择性磺化氧化。
DOI：
--
发表时间：
1992
期刊：
Drug metabolism and disposition: the biological fate of chemicals
影响因子：
--
作者：
Sadeque,AJ;Eddy,AC;Meier,GP;Rettie,AE
通讯作者：
Rettie,AE