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Molecular organization of mitochondrial quinol-fumarate reductase and its role in oxygen adaptation.

线粒体喹啉富马酸还原酶的分子组织及其在氧适应中的作用。

基本信息

批准号：
11470065
负责人：
KITA Kiyoshi
金额：
$ 8.45万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Complex II of adult Ascaris suum muscle exhibits high quinol-fumarate reductase(QFR)activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval(L3)complex II shows a much lower QFR activity than the adult enzyme, and functions as succinate-ubiquinone reductase(SQR)in aerobic respiration. We have reported the stage-specific isoforms of complex II in A.suum mitochondria, and showed that at least the flavoprotein subunit(Fp)and the small subunit of cytochrome b(cybS)of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit(Ip)of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A.suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cystein-rich regions that are thought to com … More prise the iron-sulfur clusters of the enzyme. The Ip from A.suum Larvae also was characterized because Northern hybridization showed that the adult Ip also is expressed in L3. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-therminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact that homology probing by RT-PCR using degenerated primers failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A.suum complex II share a common Ip subunit, even though the adult enzyme functions as a QFR, while larval enzyme acts as an SQR.In addition, we found novel compound, nafuredin, from Aspergillus niger. Nafuredin inhibits NADH-fumarate reductase(complexes I+II)activity, a unique anaerobic electron transport system in hilminth mitochondria, at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials using sheep. Thus, our study indicates that mitochondrial complex I is a promising target for chemotherapy and that nafuredin is a new potential lead compound as a novel anthelmintic isolated from microorganisms. Less

猪蛔虫成虫肌肉复合体II具有较高的喹酚-富马酸还原酶(QFR)活性，在其微氧环境适应过程中，在厌氧电子传递中起着关键作用。相反，幼虫(L3)复合体II表现出比成虫酶低得多的QFR活性，并且在有氧呼吸中起琥珀酸泛醌还原酶(SQR)的作用。我们已经报道了猪链霉菌线粒体中复合体II的阶段特异性亚型，并表明幼虫复合体II的黄蛋白亚基(FP)和细胞色素b小亚基(CybS)至少与成虫的不同。在本研究中，从猪链霉菌和自由生活的线虫线虫中克隆并测序了复合体II的铁硫亚单位(Ip)的完整cDNA，该亚基与FP一起形成了复合体II的催化部分。这两种线虫Ip亚基的氨基酸序列是相似的，特别是在被认为是COM…的三个富含半胱氨酸的区域附近更有价值的是这种酶的铁-硫簇。由于Northern杂交表明成虫的Ip也在L3中表达，因此也对猪链霉菌幼虫的Ip进行了鉴定。幼虫复合体II的Ip可被抗成虫Ip的抗体识别，但通过肽图谱无法与成虫Ip区分。经DEAE-Cellofine柱层析纯化的幼虫复合体II的Ip的N-末端42个氨基酸序列与成虫的成熟Ip完全相同。此外，用PVDF膜进行微量分析，幼虫和成虫的氨基酸组成基本相同。这些结果，再加上用简并引物进行同源性检测未能找到幼虫特异的Ip的事实，表明猪链霉菌复合体II的两个不同阶段特异的Ip亚基共享一个共同的Ip亚基，尽管成虫酶起QFR的作用，而幼虫的酶起SQR的作用。此外，我们从黑曲霉中发现了新的化合物nafuredin。Nafuredin抑制NADH-富马酸还原酶(复合体I+II)的活性，NADH-富马酸还原酶(复合体I+II)是一种独特的无氧电子传递系统，在芽线粒体中是一种独特的NM级。它竞争络合物I中的苯醌结合部位，并对蠕虫酶表现出高度的选择性毒性。此外，在绵羊体内试验中，那呋喃甲素对扭曲血吸虫具有驱虫活性。因此，我们的研究表明线粒体复合体I是一个很有希望的化疗靶点，而那呋喃甲素是一种新的潜在的先导化合物，作为一种从微生物中分离出来的新型驱虫剂。较少