| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
1.Proteomic analyses of mitochondrial proteins of Ascaris suum adult body-wall and free-living nematodes, Caenorhabditis elegans.
(1)We have developed the procedures to highly purify mitochondria from body wall of A.suum adult nematodes, which reside under low-oxygen tensions in host small intestine, and from C.elegans nematodes, which dwell under aerobic conditions of the atmosphere.
(2)Comparative analyses of mitochondrial proteins from A.suum body-wall and C.elegans using two-dimensional PAGE revealed remarkable differences in their protein compositions, especially those having molecular mass of around 45 and 31 kDa. Eighteen major proteins of C.elegans, developed in the two-dimensinal PAGE, were analyzed with mass spectrometer after in-gel digestion. Among them, 14 proteins were confirmed to be derived from C.elegans nematodes. At present, 11 proteins of the nematode origin were identified to be mitochondrial proteins. They include the subunit of ATP synthase (atp-2), the heat-shock
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protein (hsp-60), α-and β-subunits of propionyl CoA carboxylase, the malate dehydrogenase and aconitase, both of which are members of TCA cycle, and glutamine dehydrogenase. A protein like polin, which is known to be localized in mitochondrial outer membrane, was detected also.
2.Molecular analyses of Ascaris suum respiratory chain complexes and oxidroreductases.
(1)We have established the expression system for Ascaris suum cytochrome b_5 in E.coli. to prepare a large quantity of the cytochrome; the precursor protein was correctly processed in the bacteria and the mature protein, which exhibits properties identical to the native protein purified from the nematode body-wall, was transported to the periplasm, from which the protein was purified by one-step ion-exchange chromatography.
(2)The CybS subunit of A.suum larval (L3) complex II was isolated from the larval mitochondria to compare with adult CybS subunit; the larval CybS was shown to be different from the adult couterpart.
3.Perspective
Although all experimental plans have not completed yet, the procedures developed in the present study to purify mitochondria and to map mitochondrial proteins will accelerate proteomic studies of Ascaris adult and larval stages. Less
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