化学修饰单克隆抗体模拟谷胱甘肽过氧化物酶

化学修饰具有底物谷胱甘肽（ＧＳＨ）结合部位的单克隆抗体（４Ａ４）,使其结合部位上的丝氨酸（Ｓｅｒ）转变成谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团硒代半胱氨酸（Ｓｅ－Ｃｙｓ）,因而产生高活力的含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）．突变的４Ａ４（ｍ４Ａ４）的ＧＰＸ活力达到了天然酶活力的１９％,并对ｍ４Ａ４的酶学性质和动力学性质进行了研究;硒代谷胱甘肽（ＧＳｅＨ）连到４Ａ４结合部位,其ＧＰＸ活力由３．８６Ｕ／μｍｏｌ提高到５９８．９Ｕ／μｍｏｌ用黄嘌呤氧化酶／次黄嘌呤为中心的心肌线粒体自由基损伤模型证明Ｓｅ－ａｂｚｙｍｅ（ｍ４Ａ４）可减轻活性氧对线粒体的损伤。     

化学修饰单克隆抗体模拟谷胱甘肽过氧化物酶

化学修饰具有底物谷胱甘肽（ＧＳＨ）结合部位的单克隆抗体（４Ａ４）使其结合部位上的丝氨酸（Ｓｅｒ）转变成谷胱甘肽过氧化物酶（ＧＰＸ）的催化基因硒代半胱氨酸（ＳｅＣｙｓ）因而产生高活力的含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）突变的４Ａ４（ｍ４Ａ４）的ＧＰＸ活力达到了天然酶活力的１９％并对ｍ４Ａ４的酶学性质和动力学性质进行了研究;硒代谷胱甘肽（ＧＳｅＨ）连到４Ａ４结合部位,其ＧＰＸ活力由３．８６Ｕ／μｍｏｌ提     

化学突变具有底物结合部位的单克隆抗体制备含硒抗体酶

开发了一种制备抗体酶的新方法。用二硝基氯苯（ＤＮＣＢ）专一地与谷胱甘肽（ＧＳＨ）的巯基反应,合成出半抗原ＧＳＨ－Ｓ－ＤＮＰ。用戊二醛将半抗原偶联到牛血清白蛋白（ＢＳＡ）上,制成全抗原。再用标准的单抗制备法获得具有ＧＳＨ结合部位的单抗（４Ａ４ＩｇＧ）。用苯甲基磺酞氟（ＰＭＳＦ）和Ｈ２Ｓｅ相继处理该单抗,则将单拉结合部位上的丝氨酸（Ｓｅｒ）突变成硒代半胱氨酸（ＳｅＣｙｓ,因而在单抗结合部位上引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团。突变后的单抗具有ＧＰＸ活性,其活力已达到天然ＧＰＸ的数量级水平。动力学行为也与天然ＧＰＸ类似。这种新的含硒抗体酶有优于ＧＰＸ的一些特点。     

酒色着色菌内膜系统光依赖性同化亚硒酸钠为硒半胱氨酸的研究

分离的酒色着色菌（Ｃｈｒｏｍａｔｉｕｍｖｉｎｏｓｕｍ）内膜系统在光照和Ｏ乙酰丝氨酸（ＯＡＳ）存在的条件下,能以３５９纳摩尔／毫克细菌叶绿素·小时（ｎｍｏｌ·ｍｇＢｃｈｌ－１·ｈ－１）的速度催化ＳｅＯ２－３合成硒半胱氨酸。用超声波处理的内膜系统,催化速度仅为处理前的１１％,加入谷胱甘肽（ＧＳＨ）和还原型辅型Ⅱ（ＮＡＤＰＨ）后,其速度增加至处理前的８８３％,该反应对光具有依赖性,进一步实验表明,纯化的谷胱甘肽还原酶,在有半胱氨合酶、ＯＡＳ和ＮＡＤＰＨ共存时,能催化ＳｅＯ２－３转化为硒半胱氨酸,表明ＳｅＯ２－３在内膜系统中能被光偶联的谷胱甘肽还原酶还原为Ｓｅ２－,然后经半胱氨酸合酶的催化作用转化为硒半胱氨酸     

在酵母体内用RNA聚合酶Ⅱ表达大肠杆菌tRNA~（Sec）基因

我们将大肠杆菌的硒代半胱氨酸ｔＲＮＡ基因（ＳｅｌＣ基因）连接到分泌型表达质粒ＰＶＴ１０２Ｕ－αＭＦＬ中，并调整好阅读框架，使蛋白翻译的终止密码子位于ＳｅｌＣ基因的下游．将该质粒转化酵母，通过ＳＤＳ－ＰＡＧＥ分析，在ＳＤ液体培养基中检测出７～８ｋｄ的蛋白条带，这与理论值是相符合的。同时，我们抽提出酵母的总ＲＮＡ用互补与该ｔＲＮＡＴＣ茎环区的２１－ｍｅｒ寡核苷酸，经５′标记后作为探讨进行Ｎｏｒｔｈｅｒｎｂｌｏｔ。结果有两条较强的条带和一条较弱的条带，较强条带中一条相当于７９０ｎｔｓ左右，另一条相当于３７０ｎｔｓ左古，根据组建的表达型质粒结构资料推测７９０ｎｔｓ左右的条带是由ＲＮＡ聚合酶ＩＩ转录的未被加工的前体；３７０ｎｔｓ左右的条带是５′端被加工而３′端未被加工的分子，较弱的条带则是相当于９０ｎｔｓ左右的成熟ｔＲＮＡ分子。因此，我们认为ＲＮＡ聚合酶ＩＩ可以转录ｔＲＮＡ基因。由于实验用酵母的３′内切核酸酶含量较低，致使带长尾巴的ｔＲＮＡ前体３′端加工速度缓慢。     

在酵母体内用RNA聚合酶II表达大肠杆菌tRNA^Sec基因

我们将大肠杆菌的硒代半胱氨酸ｔＲＮＡ基因（ＳｅｌＣ基因）连接到分泌型表达质粒ＰＶＴ１０２Ｕ－αＭＦＬ中,并调整好阅读框架,使蛋白翻译的终止密码子位于ＳｅｌＣ基因的下游。将该质粒转化酵母,通过ＳＤＳ－ＰＡＧＥ分析,在ＳＤ液体培养基中检测出７￣８ｋｄ的蛋白条带,这与理论值是相符合的。同时,我们抽提出酵母的总ＲＮＡ用互补与该ｔＲＮＡ ＴψＣ茎环区的２１－ｍｅｒ寡核苷酸,经５＇标记后作为探针进行Ｎｏｒｔｈ     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

三草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＢＭ９６０２产生的中性内切β－甘露聚糖酶（ｅｎｄｏ－β－１,４－Ｄ－ｍａｎｎａｎ ｍａｎｎａｎｏｈｙｄｒｏｌａｅｓ,ＥＣ,３．２．１．７８）经硫酸铵分级沉淀、ＤＥＡＥ－纤维素（ＤＥ２２）离子交换柱层析,得到电泳纯的样品,提纯了４５．５倍,收率为５．９％。用ＳＤＳ－ＰＡＧＥ测得该酶的分子量为３５ｋＤ。用ＰＡＧＥＩＥＦ测得其等电点ｐⅠ为４．５。酶反应的     

拟南芥吲哚—3—甘油磷酸合酶免疫分析

吲哚３甘油磷酸合酶（ＩＧＳ,ｉｎｄｏｌｅ３ｇｌｙｃｅｒｏｌｐｈｏｓｐｈａｔｅｓｙｎｔｈａｓｅ,ＥＣ４．１．１．４８）在色氨酸与吲哚乙酸的生物合成途径中,催化生成吲哚３甘油磷酸。研究该基因的表达调控,对于阐明高等植物是如何调控色氨酸及生长素合成是十分重要的。利用已克隆的ＩＧＳｃＤＮＡ,构建了谷胱甘肽转移酶（ＧＳＴ,ｇｌｕｔａｔｈｉｏｎｅＳｔｒａｎｓｆｅｒａｓｅ,ＥＣ２．５．１．１８）与吲哚３甘油磷酸合酶融合蛋白的表达质粒,并将其导入到在异丙基βＤ硫代半乳糖苷（ＩＰＴＧ）诱导下能高效表达的ＩＧＳ基因缺陷菌株ｔｒｐＣ９８００λＫＣ大肠杆菌中。高表达的融合蛋白通过谷胱甘肽琼脂糖（ｇｌｕｔａｔｈｉｏｎｅａｇａｒｏｓｅ）亲和层析和ＳＤＳ聚丙烯酰胺凝胶电泳纯化后,用以免疫兔子制备抗血清。免疫印迹法分析表明拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）四种常用生态型只合成一种分子量约为４０ｋＤ的吲哚３甘油磷酸合酶蛋白。在Ａｇ＋、紫外线等逆境条件下,ＩＧＳ含量都有较大幅度的增加,这说明ＩＧＳ可能与植物的防御反应紧密相关。     

血管活性肠肽对兔支气管上皮细胞抗臭氧损伤的保护作用

用支气管刷洗法收集新西兰兔支气管上皮细胞（ＢＥＣ）,以臭氧（Ｏ３）攻击培养的ＢＥＣ,建立细胞损伤模型。测定ＢＥＣ的３Ｈ释放率计算Ｏ３的细胞毒指数（ＣＩ）、测定细胞内丙二醛（ＭＤＡ）的含量反映细胞氧化性损伤的程度,测定细胞内过氧化氢酶（ＣＡＴ）活性及还原型和氧化型谷胱甘肽（ＧＳＨ和ＧＳＳＧ）的含量反映细胞抗氧化能力。观察血管活性肠肽（ＶＩＰ）预处理对ＢＥＣ的细胞保护作用并初步探讨其保护机制。观察到：ＢＥＣ的３Ｈ释放率与Ｏ３暴露时间成正比;Ｏ３暴露２ｈ使ＭＤＡ含量和ＧＳＳＧ含量明显增加,ＧＳＨ减少;ＶＩＰ预处理呈剂量依赖性降低Ｏ３暴露的ＣＩ值、降低ＭＤＡ和ＧＳＳＧ含量、增加ＧＳＨ及ＧＳＨ／ＧＳＳＧ比值、增加ＣＡＴ活性,显示出细胞保护效应;ＶＩＰ的保护效应可被放线菌素Ｄ（Ａ－Ｄ）或蛋白激酶Ｃ阻断剂Ｈ７部分取消。结果表明：Ｏ３暴露会导致ＢＥＣ损伤,ＶＩＰ可通过增强ＢＥＣ的抗氧化能力而保护ＢＥＣ,ＶＩＰ的信号在细胞内的转导途径与基因转录及依赖ＰＫＣ的酶蛋白磷酸化有关。     

MALDI-TOF-MS对寡核苷酸的序列分析

通过对几种基质测定含不同碱基的寡核苷酸的灵敏度及精确度的比较,发现用混合基质α－氰基４－羟基肉桂酸（α－Ｃｙａｎｏ）／３－羟基吡啶羧酸（３ＨＰＡ）用于基质辅助激光解吸附电离飞行时间质谱中测定脱氧寡核苷酸,不仅能得到较好的分子离子峰,而且一些金属离子的加合物峰能得到有效的抑制,提高了测定的灵敏度。用３′－和５′－外切酶对脱氧寡核苷酸１２－ｍｅｒ（５′－ＡＴＧＣＡＴＡＴＧＣＡＴ－３′）进行部分降解,再进行ＭＡＬＤＩ－ＴＯＦ－ＭＳ分析,得到了完整的寡核苷酸的序列。     

A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs.

In eukaryotes, co-translational insertion of selenocysteine into selenoproteins necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs. We report a detailed experimental study of the secondary structures of the SECIS elements of three selenoprotein mRNAs, the rat and human type I iodothyronine deiodinase (5'DI) and rat glutathione peroxidase (GPx). Based on RNase and chemical probing, a new secondary structure model is established. It is characterized by a stem-loop structure, comprising two helices (I and II) separated by an internal loop, with an apical loop surmounting helix II. Sequence comparisons of 20 SECIS elements, arising from 2 5'DI, 13 GPx, 2 selenoprotein P, and 1 selenoprotein W mRNAs, confirm the secondary structure model. The most striking finding of the experimental study concerns a set of conserved sequences in helix II that interact to form a novel RNA structural motif consisting of a quartet composed of non-Watson-Crick base pairs 5'UGAY3': 5'UGAU3'. The potential for forming the quartet is preserved in 15 SECIS elements, but three consecutive non-Watson-Crick base pairs can nevertheless form in the other five SECIS, the central G.A tandem being invariant in all cases. A 3D model, derived by computer modeling with the use of the solution data, suggests that the base pairing interactions in the G.A tandem are of the type found in GNRA loops. The 3D model displays the quartet lying in an accessible position at the foot of helix II, which is bent at the internal loop, suggesting that the non-Watson-Crick base pair arrangement provides an unusual pattern of chemical groups for putative ligand interaction.     

A protein binds the selenocysteine insertion element in the 3'-UTR of mammalian selenoprotein mRNAs.

Several gene products are involved in co-translational insertion of selenocysteine by the tRNA(Sec). In addition, a stem-loop structure in the mRNAs coding for selenoproteins is essential to mediate the selection of the proper selenocysteine UGA codon. Interestingly, in eukaryotic selenoprotein mRNAs, this stem-loop structure, the selenocysteine insertion sequence (SECIS) element, resides in the 3'-untranslated region, far downstream of the UGA codon. In view of unravelling the underlying complex mechanism, we have attempted to detect RNA-binding proteins with specificity for the SECIS element. Using mobility shift assays, we could show that a protein, present in different types of mammalian cell extracts, possesses the capacity of binding the SECIS element of the selenoprotein glutathione peroxidase (GPx) mRNA. We have termed this protein SBP, for Secis Binding Protein. Competition experiments attested that the binding is highly specific and UV cross-linking indicated that the protein has an apparent molecular weight in the range of 60-65 kDa. Finally, some data suggest that the SECIS elements in the mRNAs of GPx and another selenoprotein, type I iodothyronine 5' deiodinase, recognize the same SBP protein. This constitutes the first report of the existence of a 3' UTR binding protein possibly involved in the eukaryotic selenocysteine insertion mechanism.     

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.     

SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3'-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins.     

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.     

The Caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence (SECIS) element that differs from mammalian SECIS elements but directs selenocysteine incorporation.

Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS elements from worm to man.     

Evidence that a polymorphism within the 3′UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer

Low selenium (Se) status has been associated with increased risk of colorectal cancer (CRC). Se is present as the amino acid selenocysteine in selenoproteins, such as the glutathione peroxidases. Se incorporation requires specific RNA structures in the 3' untranslated region (3'UTR) of the selenoprotein mRNAs. A single nucleotide polymorphism (SNP) occurs at nucleotide 718 (within the 3'UTR) in the glutathione peroxidase 4 gene. In the present study, Caco-2 cells were transfected with constructs in which type 1 iodothyronine deiodinase coding region was linked to the GPx4 3'UTR with either C or T variant at position 718. Higher reporter activity was observed in cells expressing the C variant compared to those expressing the T variant, under either Se-adequate or Se-deficient conditions. In addition, a disease association study was carried out in cohorts of patients with either adenomatous polyps, colorectal adenocarcinomas and in healthy controls. A higher proportion of individuals with CC genotype at the GPx4 T/C 718 SNP was present in the cancer group, but not in the polyp group, compared with the control group (P < 0.05). The present data demonstrate the functionality of the GPx4 T/C 718 SNP and suggest that T genotype is associated with lower risk of CRC.     

New mammalian selenocysteine-containing proteins identified with an algorithm that searches for selenocysteine insertion sequence elements

Mammalian selenium-containing proteins identified thus far contain selenium in the form of a selenocysteine residue encoded by UGA. These proteins lack common amino acid sequence motifs, but 3'-untranslated regions of selenoprotein genes contain a common stem-loop structure, selenocysteine insertion sequence (SECIS) element, that is necessary for decoding UGA as selenocysteine rather than a stop signal. We describe here a computer program, SECISearch, that identifies mammalian selenoprotein genes by recognizing SECIS elements on the basis of their primary and secondary structures and free energy requirements. When SECISearch was applied to search human dbEST, two new mammalian selenoproteins, designated SelT and SelR, were identified. We determined their cDNA sequences and expressed them in a monkey cell line as fusion proteins with a green fluorescent protein. Incorporation of selenium into new proteins was confirmed by metabolic labeling with (75)Se, and expression of SelT was additionally documented in immunoblot assays. SelT and SelR did not have homology to previously characterized proteins, but their putative homologs were detected in various organisms. SelR homologs were present in every organism characterized by complete genome sequencing. The data suggest applicability of SECISearch for identification of new selenoprotein genes in nucleotide data bases.     

Selenocysteine insertion directed by the 3'-UTR SECIS element in Escherichia coli

Co-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3′-untranslated region (3′-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria.