活性氧对大豆下胚轴线粒体结构与功能的损伤

采用百草枯、H_2O_2和Fenton反应作为外源活性氧,探讨了活性氧对大豆下胚轴离体线粒体的部分功能与结构的伤害。结果表明,活性氧O_2~-,H_2O_2和·OH都能引起线粒体结构的迅速膨胀,呼吸控制比率和氧化磷酸化效率降低,以及细胞色素氧化酶活力下降。通过对线粒体膜脂氢过氧化物和脂质共轭二烯的吸收光谱分析,阐明了活性氧对线粒体的损伤是与膜脂过氧化作用密切相关的。     

几种外源因子对大豆幼苗SOD活性的影响

高氧能促使大豆幼苗细胞内产生O_2~+速率增加,同时又使幼苗内SOD活性水平提高,以减轻O_2~+增加所引起的细胞伤害。高氧诱发O_2~+同时发生在叶绿体、线粒体和细胞溶质中,与细胞呼吸水平无明显相关。棓酸丙酯有清除体内O_2~+的能力,当浓度在1μmol/L时,能减轻大豆幼苗的氧伤害。相反,DDC是SOD的有效抑制剂,当它的浓度大于5 mmol/L时,显著抑制大豆幼苗SOD活性,增加了体内O_2~+的积累,影响了幼苗的正常生长以及幼苗氧伤害的加剧。     

线粒体氧应激损伤的防御体系

线粒体在细胞代谢和能量过程扮演着重要的角色 ,由于线粒体内膜的高选择透过性 ,线粒体较独立于外界环境 ,线粒体只有在能量吸收和转换过程中 ,受到外界环境的影响 ,产生线粒体氧应激损伤。电子由ＮＡＤＨ或ＦＡＤＨ2 通过电子传递体传递给分子氧的呼吸作用 ,也是作为呼吸副产品的活性氧(ＲＯＳ)和自由基的产生过程 ,分子氧一方面是电子传递链上电子和质子氢的末端受体 ,另一方面分子氧能启动氧化过程 ,质子氧接受一个泄漏的电子 ,变成超氧阴离子 (Ｏ·-2 ) ,Ｏ·-2是体内活性氧的主要来源 ,活性氧的不断积累能导致线粒体结构和功能的广泛损…     

乙烯促进线粒体呼吸过程中活性氧的作用

外源乙烯能促使绿豆（PhaseolusradiatusL．）黄化幼苗下胚轴线粒体呼吸高峰提前出现及峰值的提高,它对抗氰途径的促进作用比对细胞色素途径的更加显著．外源乙烯作用下绿豆线粒体的超氧阴离子自由基产生速率迅速提高,同时外源的加入使线粒体呼吸高峰的出现明显提前,这从一个侧面表明乙烯对线粒体呼吸的促进作用可能是通过提高线粒体的产生速率而实现的．SOD活性随乙烯处理时间延长而下降,但乙烯作用下的产生并非仅由SOD的活性所决定．     

毒黄素对线粒体呼吸耗氧的影响

毒黄素抑制线粒体的呼吸耗氧和氧化磷酸化活力,低浓度毒黄素下耗氧并不增加,也无过氧化氢的形成,此种效应在不同呼吸底物下是类似的。因此认为毒黄素对线粒体的抑制和微生物不同,其主要途径不是因递氢而产生过氧化氢。     

线粒体的分子进化与氧效应

线粒体通常被认为是消耗氧气产生ATP的细胞器.但自然界有多种生物具有厌氧型线粒体,其厌氧生物化学和遗传学研究表明,线粒体可能来源于兼性厌氧的α-蛋白细菌,在有氧环境中,起始共生体的厌氧功能丧失或被改变而进化成为经典的线粒体,但在厌氧环境中,有氧呼吸功能丧失了进化.厌氧型线粒体为了完成能量的转化,改变了呼吸链的组成,表现出产能模式的多样性.而经典线粒体在利用氧化反应获得能量的同时,也通过电子漏产生了自由基,对生命体本身构成了威胁.事实上,生命体呼吸链的进化是沿着不断加强对氧的利用效率和不断克服氧毒性的方向发展的.     

超氧物歧化酶在植物细胞内的分布

大豆幼苗下胚轴的SOD活性主要存在于细胞溶质,约占细胞内总活性的87.3％,其次分布在线粒体,约占总活性的6.8～7.2％。细胞溶质的SOD以Cu-Zn-SOD(SODb_1b_2b_2)类型为主,它在细胞溶质中约占86％。线粒体的SOD主要是Mn-SOD(SOD_a)类型,它在线粒体中约占74～76％。大豆幼苗下胚轴的SOD同工酶活性,SOD_a(Mn-SOD)约占13％,SODb_1b_2b_2(Cu-Zn-SOD)约占77％,SODc_1c_2c_2(Cu-Sn-SOD)约占10％,表明大豆幼苗下胚轴的三组同工酶以SODb_1b_1b_2为最强。比较绿色与黄化花生幼苗子叶SODc_1c_2c_2的差异,证明SODc_1c_2c_2的形成与光照下叶绿体的正常发育有关。     

线粒体呼吸链与活性氧

已知有氧真核生物细胞吸收的氧分子绝大部分都是在线粒体呼吸链末端细胞色素氧化酶上通过四步单电子还原生成水。但同时也有1％-2％的氧可在呼吸链中途接受单电子或双电子被部分还原生成超氧（O2·^-和过氧化氢（H2O2）作为呼吸作用的正常代谢产物。此种来源于线粒体呼吸链的O2·^-和H2O2不但在多种病理的氧化损伤中起关键作用,同样它们也是正常生理条件下对多种细胞过程具有基本调控意义的氧还信号。基于Chance实验室约自20世纪70到90年代的早期研究贡献以及20世纪90年代后其他各实验室的研究新进展,我们聚焦于下述四个相关问题的评述和讨论：（1）由于线粒体内膜面积及其含有的呼吸链复合体酶活力远远高出细胞中所有膜系数量和相关酶活力之总和,因而线粒体呼吸链产生的O2·^-和H2O2构成生物体内最大数量ROS的恒定来源;（2）线粒体呼吸链复合体III的Q循环中Qo位点中半醌自由基（UQH·）已明确是O2·^-的单电子来源;还原细胞色素C-P66^SHC是生成H2O2的双电子供体。虽然复合体I也是产生线粒体基质内O2·^-的主要来源,但由于其确切生成位点尚未明确,在invivo条件下能否产生大量O2·^-也尚有争议;（3）线粒体呼吸链产生O2·^-后的分配和跨膜转移涉及其生理病理作用机制和作用靶点等复杂而重要的问题,直到目前尚未意见一致。“质子和O2·^-循环双回路解偶联模型”整合了目前提出的几种假说的联系点,指出H^＋和O2·^-相互作用生成HO2·及其跨膜很可能是这一复杂问题的中心环节,并与O2·^-对“脂肪酸shuttling model”或O2·^-对“UCPS激活”模型形成了内在的联系;（4）线粒体呼吸形成的△P（△ψ和△pH）能直接控制呼吸链的ROS生成,并以非线性（非欧姆）相关方式通过影响Q循环中的Qo半醌的氧还态和寿命来调节O2·^-生成的急速?     

人粒细胞吞噬功能与活性氧的关系研究 Ⅱ.活性氧清除剂对吞噬发光的影响

粒细胞吞食并杀灭外来细菌的吞噬作用,在代谢上表现为葡萄糖通过己精单磷酸支路(HMP Shunt)的氧化代谢和非线粒体系统氧消耗剧增。近年来的深入研究表明,这种代谢上的变化是由于膜上结合的NADPH:O_2氧化还原酶系被激活的结果,酶使得分子氧单价还原,产生一系列活性氧物质,这些物质具有杀灭外来细菌的功能。所谓活性氧主要是指超氧化物阴离子自由基     

大豆下胚轴线粒体的衰老与膜脂的过氧化作用

离体的大豆下胚轴线粒体,在人工衰老条件下,产生了结构膨胀和细胞色素氧化酶活性的下降。衰老的线粒体也发生膜脂的过氧化作用——丙二醛、脂质的氢过氧化物和荧光脂褐色素明显增加。而且,线粒体衰老时产生的膜脂过氧化产物雨二醛,可能是膜脂的磷脂酰胆碱和磷脂酰乙醇胺中的亚麻酸发生过氧化反应的结果。     

Formation of hydrogen peroxide in the mitochondria of skeletal muscles

The generation of H2O2 in skeletal muscle mitochondria during the oxidation of NAD-dependent substrates and succinate is initiated by antimycin A but not by rotenone, which points to H2O2 formation at the respiratory chain site between the rotenone and antimycin blocks. The O2-/H2O2 ratio for alpha-ketoglutarate and succinate oxidation is approximately 1.4, which suggests that in skeletal muscle mitochondria H2O2 is predominantly formed via the superoxide radical generation. Heart and skeletal muscle mitochondria appeared to have the similar values of Vmax for H2O2 production; the catalase activity in skeletal muscle mitochondria is much lower.     

Generation of superoxide radicals by heart mitochondria: study by spin trapping under continuous oxygenation

The generation of superoxide radicals by isolated rat heart mitochondria was studied by the spin trapping technique. The sample was placed into the cavity of an EPR spectrometer in a thin-wall teflon capillary tube, which made it possible to maintain the partial oxygen pressure in the mitochondrial suspension at a constant level. Tiron was used as a spin trap, and the intensity of its EPR signal corresponded to the rate of O2-. formation in the sample. The addition of oxidation substrates (succinate, glutamate, and malate) into the incubation mixture caused the appearance of the Tiron EPR signal. The rate of superoxide radical generation by heart mitochondria strongly increased in the presence of antimycin A, an inhibitor of the Q-cycle in complex III of the respiratory chain, but it was completely depressed by another inhibitor of Q-cycle myxothiazol. The inhibition of the reverse electron transport in complex I of the respiratory chain by rotenone (oxidation substrate--succinate) caused a substantial decrease in the rate of O2-. formation by mitochondria.     

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Production of oxygen free radicals by cardiac mitochondria: effect of hypoxia-reoxygenation

The effect of the duration of hypoxia on superoxide radical production in isolated rat heart mitochondria was studied by the spin trapping technique. 4,5-Dioxybenzene was used as a spin trap. Samples were placed into the cavity of an EPR spectrometer in thin-wall gas-permeable capillary tubes, which allowed keeping the suspension of mitochondria in aerobic or hypoxic conditions. Previously we have demonstrated that the rate of superoxide generation by mitochondria isolated from postischemic hearts depends radically on the duration of myocardial ischemia. By contrast, in mitochondria isolated from intact hearts, the effect did not depend on the duration of hypoxia. The rate of superoxide production by isolated mitochondria in the presence of antimycin A (a complex III Q-cycle inhibitor) and complex I or complex II substrates was 0.9 +/- 0.1 nmole O2*- /min/mg protein at 25 degrees C. Under reoxygenation conditions, after 10 min of hypoxia, the rate of superoxide production was considerably higher than before hypoxia. At the same time, after prolonged hypoxia, its value was practically the same as after 10-min hypoxia. The results enable the conclusion that isolated mitochondria are less sensitive to hypoxic conditions than mitochondria in ischemic heart.     

Formation of superoxide radicals in isolated cardiac mitochondria: effect of adriamycin

The effect of adriamycin (doxorubicin) on superoxide radical formation in isolated rat heart mitochondria was studied by the spin trapping technique. The samples were placed into the cavity of EPR spectrometer in thin - wall gas - permeable capillary tubes, which allowed keeping the mitochondria of suspension in aerobic conditions. TIRON was used as a spin trap. We demonstrated that the rate of superoxide generation by isolated mitochondria depended radically on the presence of 1-150 microM adriamycin in incubation medium and was considerably higher than in control. The effect of adriamycin could be observed in the presence of both complex I (succinate) or complex II (glutamate and malate) substrates. The results obtained let to conclude that isolated cardiac mitochondria modified by adriamycin have a higher rate of production of superoxide radicals, which can react with spin traps not penetrating through the internal membrane.     

Regulation of nonphosphorylating electron transport pathways in soybean cotyledon mitochondria and its implications for fat metabolism

The respiration of mitochondria isolated from germinating soybean cotyledons was strongly resistant to antimycin and KCN. This oxygen uptake was not related to lipoxygenase which was not detectable in purified mitochondria. The antimycin-resistant rate of O₂ uptake was greatest with succinate as substrate and least with exogenous NADH. Succinate was the only single substrate whose oxidation was inhibited by salicyl hydroxamic acid alone, indicating engagement of the alternative oxidase. Concurrent oxidation of two or three substrates led to greater involvement of the alternative oxidase. Despite substantial rotenone-resistant O₂ uptake with NAD-linked substrates, respiratory control was observed in the presence of antimycin, indicating restriction of electron flow through complex I. Addition of succinate to mitochondria oxidizing NAD-linked substrates in state four stimulated O₂ uptake substantially, largely by engaging the alternative oxidase. We suggest that these properties of soybean cotyledon mitochondria would enable succinate received from the glyoxysome during lipid metabolism to be rapidly oxidized, even under a high cytosolic energy charge.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H₂DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Role of mitochondrial thiols of different localization in the generation of reactive oxygen species

The role of thiols of the outer and the inner membranes of mitochondria in the regulation of generation of reactive oxygen species (ROS) has been studied. It was found that N-ethylmaleimide (NEM), which penetrates through the mitochondrial membrane and binds thiols to form thioesters, at concentrations from 20 to 250 μM activates the production of superoxide anion and hydrogen peroxide during the oxidation of the substrates of complexes I and II of the respiratory chain. 5′,5′-Dithiobis-(2-nitrobenzoate) (DTNB), which does not penetrate into mitochondria and binds thiols to form disulfides, weakly activates hydrogen peroxide production during the oxidation of NAD-dependent substrates and inhibits the ROS production upon succinate oxidation. DTNB is particularly effective in inhibiting the menadione-induced formation of ROS. The differences in the ROS formation by these reagents are explained by the fact that they influence different thiol-containing proteins and enzymes. As distinct from NEM, which inhibits complex I of the respiratory chain, DTNB has no effect on the respiratory chain of mitochondria but can bind the SH-groups of NADH-quinone oxidoreductase, which is localized in the outer mitochondrial membrane and participates in the redox cycle of menadione. It was also shown that the ability to inhibit the ADP-stimulated respiration, a feature inherent in both reagents, does not significantly contribute to ROS production.     

Characterization of superoxide-producing sites in isolated brain mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.