Venkatesh K, Levi P E, Hodgson E
Department of Toxicology, North Carolina State University, Raleigh 27695.
Biochem Pharmacol. 1991 Sep 12;42(7):1411-20. doi: 10.1016/0006-2952(91)90453-c.
Flavin-containing monooxygenase (FMO; EC 1.14.13.8) was purified from mouse kidney microsomes and compared to that isolated from mouse liver microsomes. The purified enzymes from kidney and liver appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 58,000 daltons. On wide range (pH 3.5 to 9.0) isoelectric focusing, FMOs from kidney and liver resolved as a single band with an isoelectric point of 8.2. The enzymes from both kidney and liver have a pH optimum of 9.2. Thiobenzamide-S-oxidation catalyzed by both enzymes was sensitive to inhibition by the competitive inhibitors thiourea and methimazole. At an n-octylamine concentration of 3 mM, thiobenzamide-S-oxidation by the kidney FMO was increased by 122% and that by the liver FMO by 148%. Km and Vmax values were determined and compared between the two tissue enzymes for xenobiotic substrates containing nucleophilic nitrogen, sulfur or phosphorus atoms. In general, for most FMO substrates, Km and Vmax values were similar between kidney and liver FMO with only a few exceptions. The Km and Vmax values for fenthion for kidney were only half of those observed for liver FMO. Fonofos was unusual in having a low Km as well as a low Vmax for both tissue enzymes. Anti-sera developed to the FMO purified from kidney and liver showed cross-reactivity with each purified enzyme as well as with a protein with the same molecular weight as the purified FMO present in both kidney and liver microsomes. These bands showed equal intensity based on an equivalent amount of protein. Analysis of kidney and liver FMO by proteolytic digestion followed by visualization of peptides by silver staining or immunoblotting showed only minor differences between the enzymes of the two tissues. The amino acid composition of both mouse kidney and liver FMO was low in methionine and histidine and rich in aspartate/asparagine, glutamate/glutamine, leucine, valine and glycine. Edman degradation of the purified mouse kidney and liver FMO provided a single amino acid sequence of the NH2-terminus. This sequence matched exactly with the cDNA-deduced sequence reported for the pig and rabbit liver beginning with the fifth amino acid and contained the highly conserved FAD-binding domain Gly-X-Gly-X-X-Gly, commonly found in a number of other FAD-binding proteins. These studies indicate that the renal and hepatic forms of FMO from mouse are similar enzymes that are immunologically related and show only a few minor differences.
含黄素单加氧酶（FMO；EC 1.14.13.8）从小鼠肾脏微粒体中纯化出来，并与从小鼠肝脏微粒体中分离得到的该酶进行比较。肾脏和肝脏纯化后的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上呈现为一条带，表观分子量为58,000道尔顿。在宽范围（pH 3.5至9.0）等电聚焦中，肾脏和肝脏的FMO分离为一条带，等电点为8.2。肾脏和肝脏的酶最适pH均为9.2。两种酶催化的硫代苯甲酰胺-S-氧化对竞争性抑制剂硫脲和甲巯咪唑的抑制敏感。在正辛胺浓度为3 mM时，肾脏FMO催化的硫代苯甲酰胺-S-氧化增加了122%，肝脏FMO增加了148%。测定并比较了两种组织的酶对含亲核氮、硫或磷原子的外源化合物底物的Km和Vmax值。一般来说，对于大多数FMO底物，肾脏和肝脏FMO的Km和Vmax值相似，只有少数例外。肾脏中倍硫磷的Km和Vmax值仅为肝脏FMO观察值的一半。地虫磷不同寻常之处在于，两种组织的酶对其Km和Vmax均较低。针对从肾脏和肝脏纯化的FMO产生的抗血清与每种纯化酶以及与肾脏和肝脏微粒体中存在的与纯化FMO分子量相同的一种蛋白质显示出交叉反应性。基于等量蛋白质，这些条带显示出相同强度。通过蛋白水解消化分析肾脏和肝脏FMO，随后通过银染或免疫印迹对肽段进行可视化，结果显示两种组织的酶之间只有微小差异。小鼠肾脏和肝脏FMO的氨基酸组成蛋氨酸和组氨酸含量低，天冬氨酸/天冬酰胺、谷氨酸/谷氨酰胺、亮氨酸、缬氨酸和甘氨酸含量丰富。纯化的小鼠肾脏和肝脏FMO的埃德曼降解提供了NH2末端的单一氨基酸序列。该序列从第五个氨基酸开始与猪和兔肝脏报道的cDNA推导序列完全匹配，并包含在许多其他FAD结合蛋白中常见的高度保守的FAD结合结构域Gly-X-Gly-X-X-Gly。这些研究表明，小鼠的肾脏和肝脏形式的FMO是相似的酶，具有免疫相关性且仅显示出一些微小差异。
