植物交替氧化酶(AOX)在大肠杆菌中优化表达

植物交替氧化酶(Alternative Oxidase,AOX)位于高等植物线粒体内膜,从细胞色素途径的辅酶Q分岔,催化4个电子还原氧分子形成水的另一终端氧化酶。分离纯化有活性的AOX比较困难。本文研究AOX原核表达,选择pFLAG-1分泌表达载体,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导AOX优化表达,pFLAG-1-AOX大肠杆菌优化表达条件为:宿主DH5α、温度37℃、细胞密度OD600=0.6、IPTG浓度0.2mmol/L,诱导后60min收获细胞;获得少量可溶的细胞外周质AOX和大量不溶的AOX,为深入研究AOX打下基础,同时为研究膜蛋白原核表达提供依据。     

解偶联蛋白4的线粒体保护作用

线粒体解偶联蛋白(UCPs)是近年来发现的线粒体膜蛋白家族中的新成员.研究表明,解偶联蛋白4(UCP4)有调节线粒体膜电位、减少氧自由基的生成、调节细胞内钙离子浓度等作用,受细胞代谢、甲状腺激素,以及儿茶酚胺等调节.UCP4主要分布于大脑皮质和海马区,可能在脑血管病、精神分裂症、变性病等线粒体易受损的疾病中起重要作用.     

斑叶阿若母花序中交替氧化酶(AOX)的分离

斑叶阿若母（Arum maculatum）开花时花序组织中线粒体通过交替氧化呼吸途径产生很高的热量。从花序线粒体中分离的蛋白质浓度约14．0mg／ml。氧电极测定的线粒体交替氧化呼吸耗氧量为32umoles／min。用渗透压法破碎线粒体,分离线粒体膜和基质。再用非离子去污剂dBC溶解线粒体膜上的交替氧化酶（AOX）。线粒体、线粒体膜和线粒体膜可溶蛋白都具有AOX活性。而线粒体基质和线粒体膜不可溶蛋白则没有AOX活性。用快速蛋白液相层析（FPLC）法纯化了AOX,其活性可达70—100％,在-70℃中保持6个月以上仍具有活性。银染显示纯化的AOX有4个不同分子量（30—32kD）的蛋白质斑点。双向电泳表明这四个蛋白质的等电点为pH6．4至pH7．4。分离出的斑叶阿若母AOX为测量其氨基酸序列和探索其空间结构创造了条件。     

线粒体解偶联蛋白UCP2的研究进展

本文综述了线粒体解偶联蛋白2（uncoupling protein2,UCP2）研究方面的进展。UCP2定位于线粒体内膜上,通过消散线粒体内膜的质子梯度调节线粒体的功能,包括线粒体内膜电位、ATP合成、呼吸链ROS产生、线粒体钙库的存储和释放等。目前,UCP2的质子漏机理并不清楚,但体内实验表明UCP2活性可被过氧化物激活。特别是近年来UCP2调控胰岛素分泌方面的研究取得了重要进展。     

线粒体内膜解偶联蛋白UCP1在大肠杆菌中的活性表达

线粒体的呼吸耗氧偶联着ATP的合成,而位于线粒体内膜上的跨膜蛋白解偶联蛋白(uncoupling protein,UCP)能够破坏这种偶联关系.在大肠杆菌中表达了有生物活性的鼠源解偶联蛋白1(rUCP1).重组rUCP1的表达导致大肠杆菌宿主细胞生长变慢;在电子显微镜下观察免疫标记的结果显示,重组rUCP1主要表达在细菌膜上;同时将rUCP1重构到脂质体中也能够测到质子转运活性.这些结果说明,真核生物UCP1能够在原核生物中表达出有生物活性的形式,且能纯化得到足量的rUCP1蛋白用于进一步的结构生物学研究.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

杂交酸模叶片线粒体交替氧化酶呼吸途径在光破坏防御中的作用

以杂交酸模(Rumex K-1)为试材,研究了不同光强下线粒体交替氧化酶呼吸途径(AOX途径)对酸模叶片光破坏的防御作用.结果表明:在200 μmol·m-2·s-1弱光下,用水杨基羟肟酸抑制AOX途径后,Rumex K-1叶片的PSⅡ实际光化学效率、光合线性电子传递速率以及光合放氧速率均显著下降,非还原性QB反应中心显著升高,加重了叶片的光抑制,而活性氧清除机制上调,避免了活性氧的过量积累,部分缓解了Rumex K-1叶片的光抑制;在800 μmol·m-2·s-1强光下,AOX途径受抑,导致Rumex K-1叶片发生严重的光抑制,而此时活性氧清除机制的上调不足以缓解活性氧过量的积累.无论在强光还是弱光下,AOX途径在Rumex K-1叶片的光破坏防御过程中都起着重要作用,而且在强光下,AOX途径对叶片的光破坏防御作用是叶绿体内其他光破坏防御途径所不能代替的.     

解偶联蛋白2对活性氧的抑制作用

线粒体在能量代谢和自由基代谢中占据十分重要的地位。电子传递过程中形成的活性氧(reactive oxygen species,ROS)履行着众多生理功能,但过多或持续存在的ROS可能与癌症、衰老、糖尿病、动脉硬化、局部缺血或再灌注损伤等的发生有关。解偶联蛋白2(uncoupling protein 2,UCP2)作为线粒体内膜质子转运家族中的一个新成员,通过解偶联作用能降低线粒体内膜电势,使活性氢产生减少。UCP2抑制ROS产生的作用日益受到关注。     

交替氧化酶在麻疯树盐胁迫响应中的作用

麻疯树耐贫瘠,能在降水量少、环境恶劣的地区种植,但麻疯树的抗逆机理至今尚未揭晓。研究了盐胁迫对麻疯树生长的影响以及交替氧化酶(AOX)在麻疯树盐胁迫响应中的作用。结果表明,麻疯树具有较强的耐盐性,200 mmol/L和400mmol/L NaCl处理对麻疯树幼苗生长影响不大,叶片叶绿素含量和细胞含水量与对照组材料相比无明显差异。1 mol/L NaCl处理时麻疯树生长减缓,气孔开度降低,但未表现出明显的胁迫损伤。盐胁迫条件下,AOX呼吸和AOX基因的转录水平明显上升。与对照组材料相比,AOX基因转录水平在200 mmol/L和400 mmol/L NaCl处理时上升近两倍,而在1 mol/L NaCl处理时上升近3倍。结果表明,AOX可能在麻疯树盐胁迫应答过程中起关键作用,与减轻植株氧化损伤有关。     

Plant Uncoupling Mitochondrial Protein and Alternative Oxidase: Energy Metabolism and Stress

Energy-dissipation in plant mitochondria can be mediated by inner membrane proteins via two processes: redox potential-dissipation or proton electrochemical potential-dissipation. Alternative oxidases (AOx) and the plant uncoupling mitochondrial proteins (PUMP) perform a type of intrinsic and extrinsic regulation of the coupling between respiration and phosphorylation, respectively. Expression analyses and functional studies on AOx and PUMP under normal and stress conditions suggest that the physiological role of both systems lies most likely in tuning up the mitochondrial energy metabolism in response of cells to stress situations. Indeed, the expression and function of these proteins in non-thermogenic tissues suggest that their primary functions are not related to heat production.     

Compartmentation of Alternative Oxidase in Plant Mitochondria

Capacity of alternative pathway mediated, CN-resistant respirationwas measured in mitochondria isolated from three plant species:Iris bulbs, potato tuber callus and Petunia cells, grown insuspension culture. Succinate, NADH or a combination of succinateand NADH were used as respiratory substrates. In all three plantspecies electrons from exogenous NADH appeared to have no accessto the alternative pathway connected with succinate mediatedrespiration and vice versa. Therefore, besides a (functional)compartmentation of Q-pools a compartmentation of alternativepathways in plant mitochondria is proposed as well. (Received October 19, 1985; Accepted January 29, 1986)     

Regulation of Thermogenesis in Plants:The Interaction of Alternative Oxidase and Plant Uncoupling Mitochondrial Protein

Thermogenesis is a process of heat production in living organisms.It is rare in plants,but it does occur in some species of angiosperm.The heat is generated via plant mitochondrial respiration.As possible involvement in thermogenesis of mitochondrial factors,alternative ox-idases(AOXs) and plant uncoupling mitochondrial proteins(PUMPs) have been well studied.AOXs and PUMPs are ubiquitously present in the inner membrane of plant mitochondria.They serve as two major energy dissipation systems that balance mit...     

Covalent and Noncovalent Dimers of the Cyanide-Resistant Alternative Oxidase Protein in Higher Plant Mitochondria and Their Relationship to Enzyme Activity

Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis.     

Stress-Induced Changes in Ubiquinone Concentration and Alternative Oxidase in Plant Mitochondria

We have investigated the influence of stress conditions such as incubation at 4°C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4°C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH₂. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.     

Alternative Oxidase and Uncoupling Protein: Thermogenesis Versus Cell Energy Balance

The physiological role of an alternative oxidase and an uncoupling protein in plant and protists is discussed in terms of thermogenesis and energy metabolism balance in the cell. It is concluded that thermogenesis is restricted not only by a lower-limit size but also by a kinetically-limited stimulation of the mitochondrial respiratory chain.     

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.     

Signals Regulating the Expression of the Nuclear Gene Encoding Alternative Oxidase of Plant Mitochondria

Suspension cells of tobacco (Nicotiana tabacum L. cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants. We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level of Aox1 mRNA and AOX protein. This led to a large increase in the capacity for AOX respiration, defined as the amount of salicylhydroxamic acid-sensitive O2 uptake by cells in the presence of potassium cyanide. The results indicate that citrate may be an important signal metabolite regulating Aox1 gene expression. A number of other treatments were also identified that rapidly induced the level of Aox1 mRNA and AOX capacity. These included short-term incubation of cells with 10 mM acetate, 2 [mu]M antimycin A, 5 mM H2O2, or 1 mM cysteine. For some of these treatments, induction of AOX occurred without an increase in cellular citrate level, indicating that other signals (possibly related to oxidative stress conditions) are also important in regulating Aox1 gene expression. The signals influencing Aox1 gene expression are discussed with regard to the potential function(s) of AOX to modulate tricarboxylic acid cycle metabolism and/or to prevent the generation of active oxygen species by the mitochondrial electron transport chain.     

Plant Uncoupling Mitochondrial Protein Activity in Mitochondria Isolated from Tomatoes at Different Stages of Ripening

In the present study we have observed a higher state of coupling in respiring mitochondriaisolated from green as compared to red tomatoes (Lycopersicon esculentum, Mill.). Greentomato mitochondria produced a membrane potential () high enough to phosphorylate ADP,whereas in red tomato mitochondria, BSA and ATP were required to restore to the levelof that obtained with green tomato mitochondria. This supports the notion that such uncouplingin red tomato mitochondria is mediated by a plant uncoupling mitochondrial protein (PUMP;cf. Vercesi et al., 1995). Nevertheless, mitochondria from both green and red tomatoes exhibitedan ATP-sensitive linoleic acid (LA)-induced decrease providing evidence that PUMP isalso present in green tomatoes. Indeed, proteoliposomes containing reconstituted green or redtomato PUMP showed LA uniport and LA-induced H⁺ transport. It is suggested that the higherconcentration of free fatty acids (PUMP substrates) in red tomatoes could explain the lowercoupling state in mitochondria isolated from these fruits.