Kita K, Takamiya S, Furushima R, Ma Y C, Suzuki H, Ozawa T, Oya H
Department of Parasitology, Juntendo University, School of Medicine, Tokyo, Japan.
Biochim Biophys Acta. 1988 Sep 14;935(2):130-40. doi: 10.1016/0005-2728(88)90210-1.
Complex II of the anaerobic respiratory chain in Ascaris muscle mitochondria showed a high fumarate reductase activity when reduced methyl viologen was used as the electron donor. The maximum activity was 49 mumol/min per mg protein, which is much higher than that of the mammalian counterpart. The mitochondria of Ascaris-fertilized eggs, which require oxygen for its development, also showed fumarate reductase activity with a specific activity intermediate between those of adult Ascaris and mammals. Antibody against the Ascaris flavoprotein subunit reacted with the mammalian counterparts, whereas those against the Ascaris iron-sulfur protein subunit did not crossreact, although the amino acid compositions of the subunits in Ascaris and bovine heart were quite similar. Cytochrome b-558 of Ascaris complex II was separated from flavoprotein and iron-sulphur protein subunits by high performance liquid chromatography with a gel permeation system in the presence of Sarkosyl. Isolated cytochrome b-558 is composed of two hydrophobic polypeptides with molecular masses of 17.2 and 12.5 kDa determined by gradient gel, which correspond to the two small subunits of complex II. Amino acid compositions of these small subunits showed little similarity with those of cytochrome b-560 of bovine heart complex II. NADH-fumarate reductase, which is the final enzyme complex in the anaerobic respiratory chain in Ascaris, was reconstituted with bovine heart complex I, Ascaris complex II and phospholipids. The maximum activity was 430 nmol/min per mg protein of complex II. Rhodoquinone was essential for this reconstitution, whereas ubiquinone showed no effect. The results clearly indicate the unique role of Ascaris complex II as fumarate reductase and the indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase system.
当以还原型甲基紫精作为电子供体时，蛔虫肌肉线粒体厌氧呼吸链的复合物II表现出较高的延胡索酸还原酶活性。最大活性为每毫克蛋白质49微摩尔/分钟，这远高于哺乳动物相应的复合物。蛔虫受精卵的线粒体在发育过程中需要氧气，其也表现出延胡索酸还原酶活性，比活性介于成年蛔虫和哺乳动物之间。针对蛔虫黄素蛋白亚基的抗体与哺乳动物的相应亚基发生反应，而针对蛔虫铁硫蛋白亚基的抗体则不发生交叉反应，尽管蛔虫和牛心亚基的氨基酸组成非常相似。在存在十二烷基肌氨酸钠的情况下，利用凝胶渗透系统通过高效液相色谱法从黄素蛋白和铁硫蛋白亚基中分离出蛔虫复合物II的细胞色素b-558。通过梯度凝胶测定，分离出的细胞色素b-558由两条疏水多肽组成，分子量分别为17.2 kDa和12.5 kDa，它们对应于复合物II的两个小亚基。这些小亚基的氨基酸组成与牛心复合物II的细胞色素b-560的氨基酸组成几乎没有相似性。NADH-延胡索酸还原酶是蛔虫厌氧呼吸链中的最终酶复合物，它由牛心复合物I、蛔虫复合物II和磷脂重构而成。最大活性为每毫克复合物II蛋白质430纳摩尔/分钟。对苯二酚醌对于这种重构至关重要，而泛醌则没有作用。结果清楚地表明蛔虫复合物II作为延胡索酸还原酶的独特作用以及对苯二酚醌作为NADH-延胡索酸还原酶系统中低电位电子载体的不可或缺性。
