Physiological function of novel Ascaris cytochrome b5 in adaptation to low-oxygen tension

新型蛔虫细胞色素b5适应低氧张力的生理功能

• 批准号: 12670241
• 负责人: TAKAMIYA Shinzaburo
• 金额: $ 2.18万
• 依托单位: JUNTENDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670241/
• 关键词: Cytochrome b5; Adaptation to low-oxygen tension; Methemoglobin reduction; Ascaris suum; シトクロムb_5; 生理機能

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 … More 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

本研究以猪蛔虫体壁细胞色素b5的生物合成、在线虫组织和细胞器中的分布及其生理功能为研究对象。免疫印迹分析表明，A.猪细胞色素b5不定位于线粒体和微粒体，而是分布于胞浆部分，并分泌到肠周液中。在免疫组化研究中，在体壁的角质层中观察到清晰而明显的染色，表明纯化的细胞色素b5来源于角质层。与细胞色素b5的胞质分布相反，催化其还原的酶，即。e. NAD（P）H-细胞色素b5还原酶在线粒体和微粒体组分中检测到，但在胞质组分中未检测到。还原酶活性也存在于肠周液中。研究可能的r ...更多信息 在生物合成或分布的前序列中，编码A.猪细胞色素b5前体蛋白（包括一个由30个氨基酸残基组成的全长前序列和成熟蛋白）和两个编码前序列截短的细胞色素b5基因（分别缺失N端18个和28个氨基酸残基）在大肠杆菌中表达。杆菌其中，只有用编码完整前体蛋白的cDNA转化的细胞显示出产生加工的细胞色素b5，其与从体壁纯化的天然蛋白难以区分。通过亚细胞分级分离证明加工后的细胞色素b5被转运到细菌周质，这表明线虫的前序列在大肠杆菌中具有功能。杆菌细胞色素b_5从A. suum ;光谱和序列分析表明，该线虫的肠周液确实含有功能性细胞色素b5，而不是缺乏血红素部分的脱辅基蛋白。细胞色素b5在体液中参与高铁血红蛋白还原酶系统。生物信息学研究表明，C.线虫具有四种细胞色素b5的同源物。少

项目成果

Amino, H.: "Stage-specific isoforms of Ascaris suum complex II : the fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit"Mol.Biochem.Parasitol. 106. 63-76 (2000)

Amino, H.：“猪蛔虫复合体 II 的阶段特异性亚型：寄生成虫的富马酸还原酶和自由生活幼虫的琥珀酸脱氢酶共享一个共同的铁硫亚基”Mol.Biochem.Parasitol。

Amino, H., Wang, H., Hirawake, H., Saruta, F., Mizuchi, D., Mineki, R., Shindo, N., Murayama, K., Takamiya, S., Aoki, T., Kojima, S., and Kita, K.: "Stage-specific isoforms of Ascaris suum complex II : the fumarate reductase of the parasitic adult and the

Amino, H.、Wang, H.、Hirawake, H.、Saruta, F.、Mizuchi, D.、Mineki, R.、Shindo, N.、Murayama, K.、Takamiya, S.、Aoki, T.、

Kita, K.: "Electron transfer Complexes in Ascaris Mitochondria"Adv.Parasitol. (in press).

Kita, K.：“蛔虫线粒体中的电子转移复合物”Adv.Parasitol。

Amino, H.: "Stage-specific isoforms of Ascaris suum complex II : the fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit"Mol. Biochem. Parasitol.. 106. 63-76 (2000)

Amino，H.：“猪蛔虫复合体 II 的阶段特异性亚型：寄生成虫的富马酸还原酶和自由生活幼虫的琥珀酸脱氢酶共享一个共同的铁硫亚基”Mol。

Kita, K., and Takamiya, s.: "Electron transfer Complexes in Ascaris Mitochondria."Adv. Parasitol. (in press).

Kita, K. 和 Takamiya, s.：“蛔虫线粒体中的电子转移复合物”。