An analysis of fumarate reductase systems in parasitic helminths and various intermediate molluscan hosts

一些寄生蠕虫和水生软体动物主要通过磷酸烯醇-丙酮酸羧酸激酶(PEPCK)-琥珀酸途径利用葡萄糖；以琥珀酸盐为最终产物的碳水化合物代谢的替代途径。该途径的最后一步是富马酸还原反应，该反应由线粒体电子传递酶催化，富马酸作为电子受体发挥作用。富马酸还原酶系统产生ATP。本项目对不同环境下生活的猪蛔虫、威氏并殖吸虫和软体动物中间宿主富马酸还原酶系统进行了比较研究，了解其生理意义。我们实验室前期的研究表明，b型细胞色素参与蛔虫和并殖吸虫成体线粒体的富马酸还原酶系统，该细胞色素可与复合体III中的b型细胞色素区分开来。在项目过程中，在建立复合体II的分离程序后，发现蛔虫的b型细胞色素b_<558>，而吸虫的b型细胞色素b_<556>，定位于蠕虫呼吸链复合体II。蛔虫细胞色素b_<558>的中点氧化还原电位（Em）为- 34 mV。牛心脏线粒体复合体II中也存在类似的b型细胞色素，但其Em更为负（-185 mV）。b型细胞色素在寄生虫复合体II中容易被琥珀酸盐还原，而在牛肉心脏复合体II中则不容易。蛔虫和吸虫的复合体II由4个亚基组成，如牛肉心亚基。高效液相色谱法研究表明，Ascaris细胞色素b_<558>对应于两个较小的亚基。复合物II最大亚基的分子性质在三种植物中相似，免疫学同源性高。然而，两个较小的亚基具有不同的性质，免疫学同源性可以忽略不计。蛔虫复合体II具有较高的富马酸还原酶活性。此外，用牛肉心脏复合体I、蛔虫复合体II和红醌重建了nadh -富马酸还原酶的活性，表明醌是nadh -富马酸还原酶系统的重要组成部分。在软体动物中间寄主的研究方面，通过在杀软体动物存在的条件下进行厌氧浸泡实验，并对有机酸进行分析，表明pepck -琥珀酸途径在钉螺和光螺体内发挥作用。少

Some parasitic helminths and aquatic molluscs utilize glucose mainly through a phosphoenol-pyruvate carboxykinase(PEPCK)-succinate pathway; an alternative pathway of the carbohydrate metabolism with succinate as one of the resulting end-products. The terminal step of the pathway is a fumaratereducing reaction, which is catalyzed by mitochondrial electron transport enzymes and where fumarate functions as an electron acceptor. The fumarate reductase system produces ATP.In this project, fumarate reductase systems of Ascaris suum, Paragonimus westermani and molluscan intermediate hosts, which inhabit in various environments, were comparatively studied to understand their physiological significance. Previous study in our laboratory showed that a type b cytochrome, which can be distiguished from b cytochromes in complex III, participated in the fumarate reductase system in Ascaris and Paragonimus adult mitochondria. In the course of the project, the b-type cytochrome, b_<558> for Ascaris or … More b_<556> for Paragonimus, was found to be localized in complex II of the worm respiratory chain after the procedure for isolation of complex II had been established. The midpoint oxidation-reduction potential (Em) of Ascaris cytochrome b_<558> was - 34 mV. A similar b-type cytochrome exists in complex II of beef heart mitochondria though its Em is more negative (-185 mV). The b-type cytochrome in parasite complex II was easily reduced by succinate but it was not the case in beef heart complex II. Complexes II, of Ascaris and Paragonimus, were composed of four subunits like beef heart one. Studies using high performance liquid chromatography showed Ascaris cytochrome b_<558> tocorrespond to the two smaller subunits. Molecular properties of the largest subunit of complex II were similar among all three species and their immunological homology was high. However, the two smaller subunits had different properties with negligible immunological homology. Ascaris complex II had a high fumarate reductase activity. Also, an NADH-fumarate reductase activity was reconstituted with beef heart complex I, Ascaris complex II and rhodoquinone, indicating the quinone to be an essential component of the NADH-fumarate reductase system. As to studies on intermediate molluscan hosts, anaerobic immersion experiments, which were performed in the presence of molluscicides and followed by analyses of organic acids, showed the PEPCK-succinate pathway to be in function in vivo in Oncomelania nosophora and Biomphalaria glabrata snails. Less