NADPH依赖性硫氧还蛋白还原酶C在植物质体中的作用

硫氧还蛋白的氧化还原调节作用在生物界中普遍存在。它能够还原目标蛋白的二硫键,而自身的活性位点则被氧化。因此,对于新的催化循环,则需要由相应的还原酶将其再次还原成活性形式。硫氧还蛋白对维持高等植物的光合效率同样具有重要意义。叶绿体中的硫氧还蛋白分别由铁氧还蛋白依赖性硫氧还蛋白还原酶和NADPH依赖性硫氧还蛋白还原酶C(NTRC)两种酶还原。NTRC的本质是一种黄素蛋白,除了具有还原酶活性外,还整合了一个硫氧还蛋白结构域,在叶绿体和淀粉体的氧化还原调节中处于核心地位。这种特殊的双功能酶在卡尔文-本森循环、氧化戊糖磷酸途径、抗过氧化、四吡咯代谢、ATP和淀粉合成、生长素和光周期调控中扮演了多重角色。本综述总结了NTRC的生理功能,并讨论了该蛋白质对植物质体氧化还原稳态的调节机制。     

玉米过氧化物还原蛋白BAS1的原核表达及其功能研究

植物过氧化物还原蛋白BAS1是巯基依赖的过氧化物酶,通过催化的Cys残基还原过氧化氢,依赖NADPH的叶绿体硫氧还蛋白还原酶保持BAS1的还原态。玉米含有两种BAS1:2-Cys PrxA和2-Cys PrxB。利用RT-PCR方法从玉米幼叶中克隆了编码成熟2-Cys PrxA的基因,并将蛋白Cys34残基突变成Ser34。SDS-PAGE显示纯化的野生型和突变体蛋白为一条主带,分子量约为23kDa;体外蛋白结合实验表明纯化的叶绿体硫氧还蛋白还原酶通过分子间二硫键结合纯化的2Cys PrxA的C34S突变体,非还原SDS-PAGE显示纯化的野生型2Cys PrxA含有分子间二硫键组成的二体,而纯化的C34S突变体呈现单体,巯基专一性标记化合物AMS修饰及活性分析表明纯化的BAS1还原态是催化还原过氧化氢所所必须的,它由硫氧还蛋白还原酶及其辅酶NADPH所催化。     

硫氧还蛋白的共价修饰

硫氧还蛋白是细胞中普遍存在的低分子量蛋白质,为生物体所必需。硫氧还蛋白、硫氧还蛋白还原酶和烟酰胺腺嘌呤二核苷磷酸组成硫氧还蛋白系统,调节细胞的氧化还原状态。硫氧还蛋白不仅维持细胞的氧化还原平衡,还具有抗凋亡及促进细胞增殖等功能。原核细胞的硫氧还蛋白仅含有两个半胱氨酸残基,真核细胞的硫氧还蛋白除了活性中心的两个半胱氨酸残基外,通常还有另外的半胱氨酸残基。这些半胱氨酸残基的共价修饰使硫氧还蛋白具有了更丰富的功能。硫氧还蛋白的共价修饰包括谷胱甘肽化、巯基氧化、亚硝基化和烷基化。     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

硫氧还蛋白系统在COPD抗氧化中的作用研究进展

硫氧还蛋白系统是由硫氧还蛋白(thioredoxin,Trx)、硫氧还蛋白还原酶(thioredoxin reductase,TrxR)和还原型辅酶Ⅱ(NADPH)组成的多功能小分子蛋白系统,广泛表达的硫氧还蛋白作为蛋白质二硫键的还原酶,它参与很多生理过程,并发挥重要生物学功能,包括调节机体的氧化还原反应、抑制细胞凋亡、调节转录因子DNA结合活性以及免疫应答等,其中一重要作用是参与调节细胞氧化还原状态以对抗氧化应激。因此在一些炎症性疾病如慢性阻塞性肺疾病、急性呼吸窘迫综合征、肺间质疾病、哮喘、肺结节病等的发生发展中扮演重要角色,本文对硫氧还蛋白系统在慢性阻塞性肺疾病中的抗氧化作用作一综述。     

地钱,肾蕨和中山柏的NADP硫氧还蛋白系统

硫氧还蛋白(Td)是一类低分子量酸性蛋白,具有二硫键(-s-s-),通过氧化还原互变来参与很多反应(周志民等1986)。Td可被NADP-硫氧还蛋白还原酶(NTR)还原:     

硫氧还蛋白-过氧化物氧还蛋白与过氧化氢构成环路参与肿瘤的发生与发展

组织细胞可经过多种途径产生氧自由基（ROS）,而肿瘤组织由于多种应激因素会产生大量ROS,其中最重要的是过氧化氢（H2O2).H2O2对细胞发挥着致损伤及亚毒性信使的双重作用,作为信使其不仅参与调节正常细胞信号通路,重要的是促进肿瘤的发生及进展. ROS作为一种应激刺激信号激活细胞内的AP-1（activator protein 1）、Nrf-2（NF-E2-related factor 2）等核转录因子,活化后的AP-1、Nrf-2会结合到硫氧还蛋白（sulfiredoxin, SRX）基因启动子上游的调控序列,促进SRX基因的表达.SRX的表达上调则影响其下游的抗氧化蛋白,即特定亚型的过氧化物氧还蛋白（peroxiredoxin, PRX）的活性状态,最终使细胞内H2O2浓度受到调节. 由SRX-PRX轴与H2O2形成1个环路,通过调节H2O2含量来参与细胞众多信号通路.本文对H2O2、SRX及PRX各自的功能进行综述,还进一步探讨三者构成的信号环路对肿瘤的调控机制,从而了解该环路在肿瘤发生发展中所发挥的作用.     

少弱精子症患者精子候选差异蛋白Trx 2、TrxR 1的验证及在少精、弱精子症中的表达研究

目的:根据TMT技术筛选少弱精子症患者精子差异蛋白的结果,选取硫氧还蛋白2(thioredoxin 2,Trx 2)、硫氧还蛋白还原酶1(thioredoxin reductase 1,TrxR 1)进行验证,探讨二者在少精、弱精和少弱精子症中的表达变化及其意义。方法:收集105例少精子症组(O组)、150例弱精子症组(A组)、50例少弱精子症组(OA组)和106例正常精液男性(N组)精液,分离出精子,对少弱精子症进行串联质谱标签(Tandem Mass Tag,TMT)技术蛋白质组学分析,根据少弱精子症组的精子差异蛋白结果选取Trx 2、TrxR 1,通过免疫荧光和免疫印迹方法检测其在O组、A组、OA组的表达情况。结果:TMT技术蛋白质组学结果显示Trx 2为上调差异蛋白(为N组的1.31倍),TrxR 1为下调差异蛋白(为N组的0.82倍)。免疫荧光和免疫印迹结果显示O组、A组、OA组Trx 2表达显著高于N组(P0.05),O组、OA组TrxR 1的表达显著低于N组(P0.05)。二者在OA组的结果与蛋白质组学结果一致。结论:Trx 2、TrxR 1可能在少精、弱精及少弱精子症的发生中起着重要的作用,并有望成为少弱精子症患者精子的候选标志物及治疗靶点。     

硫氧还蛋白还原酶缺陷的大肠杆菌宿主促进含半胱氨酸残基的重组蛋白可溶性表达

以人工设计的,不含半胱氨氨酸残基的三元蛋白,六聚和八聚鲑鱼降钙素融合蛋白和人尿激酶原等不同半胱氨酸残基含量的外源蛋白质为例,利用大肠杆菌硫氧还蛋白还原酶基因缺陷菌GH980（DE3 trxB^-）,探索把以包涵体形式表达的外源蛋白质变为可溶性表达的可能性及其规律。研究表明：由于硫氧还蛋白还原酶基因的缺陷所引超的细胞质氧化还原态势的变化,使一些在普通大肠杆菌宿主中以包涵 形式表达,含有半胱氨酸残基的重组蛋白,在GJ980中能在一定程度上以可溶性蛋白质形式表达;不含有半胱氨酸残基的重组蛋白在GJ980中仍以包涵体形式表达,推测重组蛋白在GJ980细胞质中形成二硫键对其正确构象的形成具有一定的作用。     

福氏志贺菌硫氧还过氧化物酶的表达、纯化、活性检测及晶体生长的初步研究

Sf2523蛋白属于硫氧还蛋白过氧化物酶(Prx)家族,在有氧代谢过程中消除活性氧,起到保护生命大分子的重要作用.通过构建原核表达体系,可溶性表达并纯化了Sf2523酶蛋白,并对蛋白进行了过氧化物酶活性检测,证明其依然具有天然活性,酶的核心结构并未发生变化.由分子排阻色谱结果发现,酶蛋白体内和体外聚集状态不同,离体蛋白聚集状态不稳定,趋于二体化.将纯化的单体蛋白即时进行了结晶实验,初筛长出了针状晶体.通过进一步切除标签蛋白进行优化处理,最终得到了均匀的三维单晶.     

Myeloperoxidase-Halide-Hydrogen Peroxide Antibacterial System

An antibacterial effect of myeloperoxidase, a halide, such as iodide, bromide, or chloride ion, and H(2)O(2) on Escherichia coli or Lactobacillus acidophilus is described. When L. acidophilus was employed, the addition of H(2)O(2) was not required; however, the protective effect of catalase suggested that, in this instance, H(2)O(2) was generated by the organisms. The antibacterial effect was largely prevented by preheating the myeloperoxidase at 80 C or greater for 10 min or by the addition of a number of inhibitors; it was most active at the most acid pH employed (5.0). Lactoperoxidase was considerably less effective than was myeloperoxidase when chloride was the halide employed. Myeloperoxidase, at high concentrations, exerted an antibacterial effect on L. acidophilus in the absence of added halide, which also was temperature- and catalase-sensitive. Peroxidase was extracted from intact guinea pig leukocytes by weak acid, and the extract with peroxidase activity had antibacterial properties which were similar, in many respects, to those of the purified preparation of myeloperoxidase. Under appropriate conditions, the antibacterial effect was increased by halides and by H(2)O(2) and was decreased by catalase, as well as by cyanide, azide, Tapazole, and thiosulfate. This suggests that, under the conditions employed, the antibacterial properties of a weak acid extract of guinea pig leukocytes is due, in part, to its peroxidase content, particularly if a halide is present in the reaction mixture. A heat-stable antibacterial agent or agents also appear to be present in the extract.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Hydrogen Peroxide Metabolism in Yeasts

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H₂O₂ generated in the oxidation of methanol by alcohol oxidase. Not only H₂O₂ generated intracellularly but also H₂O₂ added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H₂O₂ consumption during growth in chemostat cultures on mixtures of glucose and H₂O₂, it appeared that the mutant was capable of decomposing H₂O₂ at a rate as high as 8 mmol · g of cells⁻¹ · h⁻¹. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H₂O₂. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H₂O₂ at a high rate when cells were grown on mixtures of glucose and H₂O₂. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H₂O₂, the level of catalase remained low, but CCP increased with increasing rates of H₂O₂ utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H₂O₂ detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.     

Hydrogen Peroxide in Human Blood

Blood and plasma of humans and rats were analyzed for hydrogen peroxide. The samples were analyzed after deproteinization with trichloroacetic acid, immediately after they were withdrawn from human volunteers or rats. A radio-isotopic technique based on peroxide-dependent decarboxylation of 1-¹⁴C-alpha-ketoacids and consequent liberation of ¹⁴CO₂ was used. The results demonstrate the presence ofmicromolar levels of H₂O₂, both, in the plasma as well as in the whole blood. The values in the whole blood were substantially greater than the plasma. This was true for rats as well as humans. The presence of such significant quantities of H₂O₂ in the blood have been demonstrated for the first time. The investigation, therefore, opens a newer avenue of research on diseases purported to be related to the generation of oxygen radicals in vivo.