Proteomic analyses of oxygen -responding proteins of Ascaris suum nematodes

猪蛔虫线虫氧响应蛋白的蛋白质组学分析

• 批准号: 14570220
• 负责人: TAKAMIYA Shinzaburo
• 金额: $ 2.56万
• 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: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570220/
• 关键词: Ascaris suum; Adaptation to low-oxygen tension; proteome; mitochondria; Sub-cellular fractionation; Cytochrome b_5

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 … More 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.PerspectiveAlthough 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

1.猪蛔虫成虫体壁线虫和自由生活线虫的线粒体蛋白质组学分析。(1)We已经开发了从猪蛔虫成虫线虫的体壁和从秀丽隐杆线虫线虫的体壁高度纯化线粒体的方法，猪蛔虫成虫线虫在宿主小肠中处于低氧张力下，秀丽隐杆线虫线虫在大气的有氧条件下。（2）猪蛔虫和秀丽隐杆线虫体壁线粒体蛋白质的双向电泳比较分析表明，二者的蛋白质组成存在显著差异，尤其是分子量在45和31 kDa左右的蛋白质。双向电泳显示线虫的18个主要蛋白质，经胶内酶切后用质谱仪分析。其中，14个蛋白被证实来自秀丽隐杆线虫。目前已鉴定出11种线虫源蛋白为线粒体蛋白。它们包括ATP合酶亚基（atp-2）、热休克蛋白（heat-shock protein）和ATP酶亚基。 ...更多信息 蛋白（hsp-60）、丙酰辅酶A羧化酶的α-和β-亚基、苹果酸脱氢酶和乌头酸酶（均为TCA循环的成员）以及谷氨酰胺脱氢酶。猪蛔虫呼吸链复合物和氧化还原酶的分子分析。(1)We建立了猪蛔虫细胞色素b_5的原核表达系统。以制备大量的细胞色素;前体蛋白在细菌中被正确地加工，并且表现出与从线虫体壁纯化的天然蛋白相同的性质的成熟蛋白被运输到周质，通过一步离子交换色谱从周质中纯化蛋白。（2）从猪蛔虫幼虫线粒体中分离出CybS亚基，并与成虫的CybS亚基进行了比较，结果表明幼虫的CybS亚基与成虫的CybS亚基存在差异。少

项目成果

Takarniya S. et al.: "Heterologous expression of Ascaris suum cytochrome b_5 precursor protein: a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coil."Archives of Biochemistr

Takarniya S. 等人：“猪蛔虫细胞色素 b_5 前体蛋白的异源表达：组氨酸标记的全长前序列被正确处理，以将成熟蛋白转运至大肠杆菌的周质。”Biochemistr 档案

Amino H. et al.: "Isolation and characterization of the stage-specific cytochrome b small subunit(CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria."Molecular & Biochemical Parasitology. 128. 175-186 (2003)

Amino H. 等人：“从幼虫线粒体的有氧呼吸链中分离和表征猪蛔虫复合体 II 的阶段特异性细胞色素 b 小亚基 (CybS)。”

Mineki R, Shindo N, (6人中5番目) et al.: "In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one-and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis."Proteomics. 2. 1672-1681 (2002)

Mineki R，Shindo N，（6 中的第 5）等人：“在一维和二维十二烷基硫酸钠聚丙烯酰胺凝胶电泳过程中用丙烯酰胺进行原位烷基化以鉴定蛋白质中的半胱氨酰残基。”蛋白质组学 2。1672- 1681 (2002)

Takamiya S. et al.: "Helerologous expression of Ascarils suum cytochrome b_5 precursor protein : a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli."Archives of Biochemist

Takamiya S. 等人：“猪蛔虫细胞色素 b_5 前体蛋白的异源表达：组氨酸标记的全长前序列被正确处理，以将成熟蛋白转运至大肠杆菌的周质。”生物化学家档案

Takamiya S., et al.: "Heterologous expression of Ascaris suum cytochrome b_5 precursor protein : a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli"Archives of Biochemistr

Takamiya S.等人：“猪蛔虫细胞色素b_5前体蛋白的异源表达：组氨酸标记的全长前序列被正确处理，以将成熟蛋白转运至大肠杆菌的周质”生物化学档案