Proteomic analyses of oxygen -responding proteins of Ascaris suum nematodes

1.猪蛔虫成虫体壁和线虫线粒体蛋白的蛋白质组学分析。(1)我们开发了从A.suum成年线虫体壁和秀丽隐杆线虫体壁高度纯化线粒体的方法，前者生活在宿主小肠的低氧紧张环境中，后者生活在有氧环境中。(2)利用二维PAGE技术对秀丽隐杆线虫和a.s suum体壁线粒体蛋白进行比较分析，发现它们的蛋白质组成存在显著差异，特别是分子质量在45和31 kDa左右的线粒体蛋白。在二维PAGE中开发的秀丽隐杆线虫的18种主要蛋白经凝胶内消化后用质谱分析。其中14个蛋白被证实来源于秀丽隐杆线虫。目前，线虫起源的11个蛋白被鉴定为线粒体蛋白。它们包括ATP合成酶亚基（ATP -2）、热休克蛋白（hsp-60）、丙酰辅酶a羧化酶α和β亚基、苹果酸脱氢酶和乌头酸酶（两者都是TCA循环的成员）和谷氨酰胺脱氢酶。我们还发现了一种类似于polin的蛋白质，已知它位于线粒体外膜。猪蛔虫呼吸链复合物和氧化还原酶的分子分析。(1)建立了猪蛔虫细胞色素b_5在大肠杆菌中的表达体系。制备大量的细胞色素；前体蛋白在细菌中被正确处理，成熟蛋白被运输到外周质，并通过一步离子交换色谱法从外周质中纯化蛋白质。成熟蛋白具有与从线虫体壁中纯化的天然蛋白相同的特性。(2)从幼鱼线粒体中分离出幼鱼（L3）复合体II的CybS亚基，并与成虫的CybS亚基进行比较；幼虫与成虫有明显的差异。虽然所有的实验计划尚未完成，但本研究中开发的纯化线粒体和绘制线粒体蛋白的程序将加速蛔虫成虫和幼虫阶段的蛋白质组学研究。少

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