Lohrer H, Krauth-Siegel R L
Institut für Biochemie II, Universität Heidelberg, Federal Republic of Germany.
Eur J Biochem. 1990 Dec 27;194(3):863-9. doi: 10.1111/j.1432-1033.1990.tb19480.x.
From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.
从恰加斯病的病原体克氏锥虫中分离出了一种硫辛酰胺脱氢酶。该酶是一种FAD-胱氨酸氧化还原酶，与克氏锥虫的锥虫硫醇还原酶（该寄生虫硫醇代谢的关键酶）具有许多物理和化学性质。1. 从60克克氏锥虫的上鞭毛体细胞中提取出了2.7毫克硫辛酰胺脱氢酶。该黄素酶被纯化了3000倍达到同质，总产率为26%。2. 该酶是一种二聚体，亚基的相对分子质量为55,000。在pH 7.0条件下，对于1毫摩尔硫辛酰胺（Km约为5毫摩尔）和100微摩尔NADH（Km = 23微摩尔），其比活性为297 U/毫克。3. 当有过量NADH时，该酶被还原为EH2.NADH复合物，加入硫辛酰胺后，它又被重新氧化，这表明它可以在氧化态E和双电子还原态EH2之间循环。4. 如对该酶进行N端测序所示，30个位置中有21个与猪心和人肝硫辛酰胺脱氢酶的相同。测序部分包含GGGPGG片段，它代表FAD焦磷酸部分的结合位点。5. 将Eox还原为双电子还原态后，该酶被亚硝基脲类药物1,3-双(2-氯乙基)-1-亚硝基脲（卡莫司汀）特异性抑制，推测是通过在一个新生的活性位点硫醇处进行氨甲酰化作用。6. 用针对克氏锥虫硫辛酰胺脱氢酶和锥虫硫醇还原酶产生的多克隆兔抗体对相应的酶具有特异性，这通过对纯蛋白和细胞提取物的免疫印迹得以证明。
