萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)是一个定位于线粒体的蛋白质.为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的RsPHGPx.用重组RsPHGPx 蛋白免疫兔子获得了抗RsPHGPx 的多克隆抗血清,以重组RsPH ...萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)是一个定位于线粒体的蛋白质.为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的RsPHGPx.用重组RsPHGPx 蛋白免疫兔子获得了抗RsPHGPx 的多克隆抗血清,以重组RsPH G Px 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗RsPHGPx 的抗体.将纯化好的抗体偶联到一个N-羟基琥珀酰亚胺(NHS)预先激活的琼脂糖柱子上,装配成一个以单特异性的抗RsPHGPx 抗体为配体的免疫亲和层析柱.经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案.按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质.免疫印迹分析表明,该蛋白质被抗RsPH G Px 的抗血清特异识别.酶活性分析表明,该蛋白质具有显著的PH G Px 活性.这些结果表明,纯化到的特异蛋白质是萝卜的RsPH G Px天然蛋白.这是首个关于定位于植物细胞器的PH G Px 蛋白纯化的报道.这一结果为准确测定RsPH G Px 信号肽的切割位点奠定了基础,并将有助于对植物PH G Px 的亚细胞定位机制及其生理功能的深入研究.展开更多
More than 20 sequences of phospholipid hydroperoxide glutathione peroxidase (PHGPX) from a sequence database were analyzed. The analyses show that the primary structures of most PHGPX proteins have three highly con...More than 20 sequences of phospholipid hydroperoxide glutathione peroxidase (PHGPX) from a sequence database were analyzed. The analyses show that the primary structures of most PHGPX proteins have three highly conserved regions forming a catalytic center and have more than 50% amino acid sequence identity in common. However, two PHGPXs from bovine and swine with the same function have very low similarity with typical PHGPXs and do not have the three highly conserved regions. Thus, the PHGPX proteins are divided into two types: those with the three highly conserved regions, designated as PHGPX I, and the others as PHGPX II. In general, type I proteins are composed of ca.170 amino acid residues; a few of them have an extra signal peptide sequence at the N terminal of the protein. The composition of plant and animal PHGPX amino acids is very different, with most plant PHGPXs being weak acidic, while most animal ones are alkaline. Another specific conservative motif is also found in plant PHGPX proteins. System evolution analysis shows that ortholog and paralog evolution models both exist in PHGPXs, with the plant PHGPX and the animal PHGPX diverging exclusively into two branches in PHGPX I. The information revealed by the evolution tree agrees with the general species evolution process from low to advanced and from simple to complicated. 展开更多
文摘萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)是一个定位于线粒体的蛋白质.为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的RsPHGPx.用重组RsPHGPx 蛋白免疫兔子获得了抗RsPHGPx 的多克隆抗血清,以重组RsPH G Px 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗RsPHGPx 的抗体.将纯化好的抗体偶联到一个N-羟基琥珀酰亚胺(NHS)预先激活的琼脂糖柱子上,装配成一个以单特异性的抗RsPHGPx 抗体为配体的免疫亲和层析柱.经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案.按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质.免疫印迹分析表明,该蛋白质被抗RsPH G Px 的抗血清特异识别.酶活性分析表明,该蛋白质具有显著的PH G Px 活性.这些结果表明,纯化到的特异蛋白质是萝卜的RsPH G Px天然蛋白.这是首个关于定位于植物细胞器的PH G Px 蛋白纯化的报道.这一结果为准确测定RsPH G Px 信号肽的切割位点奠定了基础,并将有助于对植物PH G Px 的亚细胞定位机制及其生理功能的深入研究.
基金Supported by the National Natural Science F oundationof China(Nos. 3 9770 0 78and 3 0 170 0 80 )the NationalTransgenic Plant Research Project(No.J99-A-041)+1 种基金State Key Basic Research and Development Plan(No.G199980 10 10 0 )Yunnan Province-Unive
文摘More than 20 sequences of phospholipid hydroperoxide glutathione peroxidase (PHGPX) from a sequence database were analyzed. The analyses show that the primary structures of most PHGPX proteins have three highly conserved regions forming a catalytic center and have more than 50% amino acid sequence identity in common. However, two PHGPXs from bovine and swine with the same function have very low similarity with typical PHGPXs and do not have the three highly conserved regions. Thus, the PHGPX proteins are divided into two types: those with the three highly conserved regions, designated as PHGPX I, and the others as PHGPX II. In general, type I proteins are composed of ca.170 amino acid residues; a few of them have an extra signal peptide sequence at the N terminal of the protein. The composition of plant and animal PHGPX amino acids is very different, with most plant PHGPXs being weak acidic, while most animal ones are alkaline. Another specific conservative motif is also found in plant PHGPX proteins. System evolution analysis shows that ortholog and paralog evolution models both exist in PHGPXs, with the plant PHGPX and the animal PHGPX diverging exclusively into two branches in PHGPX I. The information revealed by the evolution tree agrees with the general species evolution process from low to advanced and from simple to complicated.
