| Co-Investigator(Kenkyū-buntansha) |
AMINO Hisako University of Tokyo, Graduate School of Medicine, Assistant Professor, 大学院・医学系研究科, 助手 (10323601)
TAKEO Satoru University of Tokyo, Graduate School of Medicine, Assistant Professor, 大学院・医学系研究科, 助手 (40302666)
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| Budget Amount *help |
¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 2000: ¥5,600,000 (Direct Cost: ¥5,600,000)
Fiscal Year 1999: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Research Abstract |
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
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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
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