| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
In the current research project, the novel cytochrome b5, previously demonstrated in Ascaris suum body wall, has been studied to elucidate its biosynthesis, distribution in the nematode's tissues and organella, and physiological functions. Immunoblotting analysis of subcellular fractions of the body wall and perienteric fluid suggested that A. suum cytochrome b5 was not localized in mitochondria nor in microsome, but distributed in cytosolic fraction, and secreted into the perienteric fluid. The clear and distinct staining was observed in the cuticle layers of the body wall in immunohistochemical studies, indicating that purified cytochrome b5 was derived from the cuticle layers. In contrast to cytosolic distribution of the cytochrome b5, enzymes catalyzing its reduction, i. e. NAD(P)H- cytochrome b5 reductase, were detected in mitochondrial and microsomal fractions, but not in the cytosolic fraction. The reductase activity was also present in the perienteric fluid. To study possible r
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oles of the presequence in biosynthesis or distribution, the gene encoding A. suum cytochrome b5 precursor protein, comprising a full-length presequence consisting of 30 amino acid residues and mature protein, and two genes encoding the cytochrome b5 with truncated presequence, one lacking 18, and the other lacking 28 amino acid residues from its N-terminus, were expressed in E. coli. Among them, only the cells transformed with cDNA encoding intact precursor protein were shown to produce processed cytochrome b5, which is indistinguishable from native protein purified from the body wall. The processed cytochrome b5 was demonstrated by subcellular fractionation to be transported to the bacterial periplasm, indicating that the nematode's presequence was functional in E. coli. Cytochrome b5 was partially purified from perienteric fluid of A. suum ; the spectral and sequence analysis showed that the nematode's perienteric fluid in deed contained functional cytochrome b5, not apo-protein lacking heme moiety. The cytochrome b5 was shown to be involved in methemoglobin reductase system in the body fluid. Bioinformatic studies showed that C. elegans possessed four homologues of cytochrome b5. Less
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