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

本项目研究了先前在蛔虫体壁中发现的新型细胞色素b5，以阐明其生物合成、在线虫组织和细胞器中的分布及其生理功能。体壁和肠周液亚细胞组分的免疫印迹分析表明，A. suum细胞色素b5不局限于线粒体和微粒体，而是分布在细胞质组分中，并分泌到肠周液中。免疫组化观察到体壁角质层染色清晰，表明纯化的细胞色素b5来源于角质层。与细胞色素b5的细胞质分布相反，催化其还原的酶，即NAD(P)H-细胞色素b5还原酶，在线粒体和微粒体部分检测到，但在细胞质部分未检测到。肠周液中也有还原酶活性。为了研究细胞色素b5前体蛋白在生物合成和分布中可能存在的更多位点，我们在大肠杆菌中表达了a . suum细胞色素b5前体蛋白编码基因，该基因包含一个由30个氨基酸残基和成熟蛋白组成的全长前序列，以及两个编码细胞色素b5前序列截断的基因，其中一个缺失18个氨基酸残基，另一个缺失28个氨基酸残基。其中，只有编码完整前体蛋白的cDNA转化的细胞产生加工细胞色素b5，与从体壁纯化的天然蛋白没有区别。亚细胞分离证实处理后的细胞色素b5可以转运到细菌的周质，这表明线虫的前序在大肠杆菌中起作用。细胞色素b5从猪肠周液中部分纯化；光谱分析和序列分析表明，线虫肠周液中确实含有功能性细胞色素b5，而不是缺乏血红素片段的载脂蛋白。细胞色素b5参与体液中高铁血红蛋白还原酶系统。生物信息学研究表明，秀丽隐杆线虫具有4种细胞色素b5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”。