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

在目前的研究项目中，以前在猪蛔虫体壁中发现的新的细胞色素b5已经被研究，以阐明它的生物合成，在线虫组织和细胞器中的分布，以及生理功能。免疫印迹分析表明，猪链霉菌细胞色素b5既不定位于线粒体，也不定位于微粒体，而分布于胞浆部分，并分泌到肠周液中。免疫组织化学染色显示，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的胞质分布相反，催化细胞色素b5还原的酶，即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…编码猪链霉菌细胞色素b5前体蛋白的基因(由30个氨基酸残基和成熟蛋白组成的全长前序列)和两个编码细胞色素b5的基因(其中一个缺失18个氨基酸，另一个在N端缺失28个氨基酸残基)在大肠杆菌中表达。其中，只有编码完整前体蛋白的细胞才能产生加工过的细胞色素b5，这与从体壁纯化的天然蛋白没有区别。经亚细胞分离后的细胞色素b5被运输到细菌周质中，表明线虫的前序列在大肠杆菌中是有功能的。从猪链霉肠周液中部分纯化出细胞色素b5，光谱分析和序列分析表明，该线虫的肠周液中含有功能性细胞色素b5，而不是缺乏血红素部分的脱辅蛋白。细胞色素b5参与了体液中的高铁血红蛋白还原酶系统。生物信息学研究表明，线虫具有四种细胞色素b5同源物。较少

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