谷胱甘肽/谷胱甘肽过氧化物酶系统在微生物细胞抗氧胁迫系统中的作用

谷胱甘肽(GSH)/谷胱甘肽过氧化物酶(GPx)系统在不同微生物细胞抵抗氧胁迫中的生理功能不尽相同。该系统在真核模式微生物酿酒酵母中是必需存在的,在维持胞内氧化还原平衡和抵抗氧胁迫中发挥主要作用。然而,在原核微生物中,该系统只是条件性的,即部分胞内存在谷胱甘肽还原酶和GPx的原核微生物,如流感嗜血杆菌和乳酸乳球菌,可通过从胞外吸收GSH,形成条件性的依赖于GSH的GPx系统,参与抵抗氧胁迫。     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生,其中包括超氧化物歧化酶(SOD,EC1.15.L1)、抗坏血酸过氧化物酶(APX,EC1.11.1.11)、过氧化氢酶(CAT,E.C.1.11.1.6)和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9).它们在清除活性氧过程中起着不同的作用.GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少.最近几年研究表明,植物体内也存在类似于哺乳动物的GPXs家族,并对其功能研究已初见端倪.本文综述了有关GPXs的结构以及植物GPXs功能的研究进展.     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生, 其中包括超氧化物歧化酶(SOD, EC1.15.1.1)、抗坏血酸过氧化物酶(APX, EC1.11.1.11)、过氧化氢酶(CAT, E.C.1.11.1.6 )和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9)。它们在清除活性氧过程中起着不同的作用。GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少。最近几年研究表明, 植物体内也存在类似于哺乳动物的GPXs家族, 并对其功能研究已初见端倪。本文综述了有关GPXs的结构以及植物GPXs功能的研究进展。     

硒,谷胱甘肽过氧化物酶和不饱和脂肪酸在鼠亚细胞中的分布

采和梯度离心和放射性同位素等方法从鼠脑中分离得到髓磷脂、突触囊、轻突触体、重突触体、线粒体６个亚细胞组分,分别测定了各亚细胞中硒－７５、谷胱甘肽过氧化物酶和不饱和脂肪酸的含量,结果表明这上结成分在鼠脑亚细胞中的分布呈明显的相关性,同时首次在突触囊、线粒体和微粒体中检测到三咱不同的谷胱甘肽过氧化物酶的活性峰,其中之一可能是红细胞谷胱甘过氧化物酶（ＥＣ１．１１．１．９）。还就机体的自我保护机制和硒在脑     

硒、谷胱甘肽过氧化物酶和不饱和脂肪酸在鼠脑亚细胞中的分布

采用梯度离心和放射性同位素等方法从鼠脑中分离得到髓磷脂、突触囊、轻突触体、重突触体、线粒体６个亚细胞组分。分别测定了各亚细胞中硒－７５、谷胱甘肽过氧化物酶和不饱和脂肪酸的含量,结果表明这些成分在鼠脑亚细胞中的分布呈现明显的相关性,同时首次在突触囊、线粒体和微粒体中检测到三种不同的谷胱甘肽过氧化物酶的活性峰,其中之一可能是红细胞谷胱甘肽过氧化物酶（ＥＣ１．１１．１．９）．还就机体的自我保护机制和硒在脑组织中的重要作用进行了讨论。     

纳米硒对肉鸡肝细胞中细胞谷胱甘肽过氧化物酶活性的影响

以亚硒酸钠和蛋氨酸硒为对照,研究了纳米单质硒(纳米硒)对肉鸡肝细胞中细胞谷胱甘肽过氧化物酶(cGPx)活性的影响。每种硒源分别以0.01、0.05、0.10、0.30、0.50、1.0μmol/L6个硒添加浓度培养肉鸡肝细胞,测定培养后0、24、48、72、96h肉鸡肝细胞cGPx活性。结果显示:亚硒酸钠添加浓度(以硒计)在0.01￣0.10μmol/L、蛋氨酸硒和纳米硒添加浓度(以硒计)在0.01￣0.30μmol/L,cGPx活性随着硒添加浓度的增加而增加;亚硒酸钠添加浓度在0.10￣1.0μmol/L、蛋氨酸硒添加浓度在0.30￣1.0μmol/L,cGPx活性随着硒添加浓度的增加而下降,而纳米硒添加浓度在0.30￣1.0μmol/L,cGPx活性始终保持在高峰平台。结果表明,3种硒源的剂量-效应关系曲线中的最适剂量范围宽度依次为:纳米硒>蛋氨酸硒>亚硒酸钠。     

高温胁迫对扶桑绵粉蚧成虫过氧化物酶和谷胱甘肽-S-转移酶活性的影响

为了研究高温胁迫对扶桑绵粉蚧的影响,将该虫分别置于35℃、38℃、41℃、44℃和47℃水浴处理2 h、3 h和4 h,然后置于26℃下恢复2 h,测定成虫的过氧化物酶(POD)和谷胱甘肽-S-转移酶(GSTs)活力的变化。结果表明在处理2 h、3 h和4 h下,对照(26℃)的POD活力值分别为(0.0527±0.0015)mmol/min、(0.0508±0.0015)mmol/min和(0.0483±0.0072)mmol/min,均高于高温各处理的POD活力值;5个温度处理下的POD活力值变化为低(35℃)-高(38℃)-低(41、44、47℃)的变化趋势;当处理2 h时,不同温度下的POD活力值分别是(0.0183±0.0009)(35℃)、(0.0480±0.0012)(38℃)、(0.0227±0.0012)(41℃)、(0.0197±0.0003)(44℃)、(0.0173±0.0007)(47℃)mmol/min。且在不同温度和时间处理下,成虫的GSTs活性变化趋势与POD活性变化趋势一致。即不同时间处理下,对照的GSTs活性均高于高温各处理的GSTs,其对照的GSTs活力值分别为(0.5537±0.0044)(2 h)、(0.5358±0.0078)(3 h)和(0.5291±0.0264)(4 h)mmol/min;5个温度处理下的GSTs活力值变化为低(35℃)-高(38℃)-低(41℃、44℃、47℃)的变化趋势;当处理3 h时,不同温度下的GSTs活力值分别是(0.5114±0.0116)(35℃)、(0.5426±0.0009)(38℃)、(0.4861±0.0073)(41℃)、(0.3657±0.0029)(44℃)、(0.3404±0.0156)mmol/min(47℃)。因此高温对扶桑绵粉蚧体内的POD和GSTs活力存在影响。     

谷胱甘肽磷脂氢过氧化物酶研究进展

谷胱甘肽磷脂氢过氧化物酶(PHGPx)是生物体内一种重要的抗氧化酶。它是一种硒依赖性蛋白,在谷胱甘肽(GSH)的参与下能特异性地还原磷脂氢过氧化物(PLOOH)和胆固醇氢过氧化物(ChOOH),从而保护生物膜免受过氧化损伤。它还是核酸等生物大分子的重要保护剂,并且在细胞凋亡调控中发挥作用。     

水分胁迫对刺槐叶和根谷胱甘肽抗氧化系统的影响

在人工控水条件下,采用土壤最大持水量70%、55%、40%的水分处理模拟环境中的正常水分、轻度和重度水分胁迫处理,测定了刺槐叶片和根系中还原型谷胱甘肽(GSH)和还原型抗坏血酸(AsA)含量以及谷胱甘肽还原酶(GR)、谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)活性,以探讨水分胁迫条件下刺槐谷胱甘肽抗氧化系统的保护作用.结果显示:各水分处理的刺槐叶片GSH和AsA含量及GR 和SOD活性均明显高于根,根中GSH-Px活性只有在重度水分胁迫处理下大于叶片.随水分胁迫加剧,刺槐GSH含量在叶片中先升高后降低,在根中不断升高;AsA含量在叶中持续降低,在根中先升高后降低;GR活性在叶片和根系中都会降低,GSH-Px和SOD活性在叶中先升高后降低,在根中均持续升高.研究表明,刺槐谷胱甘肽抗氧化系统的GSH和GSH-Px对干旱胁迫诱发的活性氧清除起主要作用,同时提高GSH含量和GSH-Px活性是刺槐应对干旱胁迫的重要措施.     

Ketogenic Diet Increases Glutathione Peroxidase Activity in Rat Hippocampus

Ketogenic diets have been used in the treatment of refractory childhood epilepsy for almost 80 years; however, we know little about the underlying biochemical basis of their action. In this study, we evaluate oxidative stress in different brain regions from Wistar rats fed a ketogenic diet. Cerebral cortex appears to have not been affected by this diet, and cerebellum presented a decrease in antioxidant capacity measured by a luminol oxidation assay without changes in antioxidant enzyme activities—glutathione peroxidase, catalase, and superoxide dismutase. In the hippocampus, however, we observed an increase in antioxidant activity accompanied by an increase of glutathione peroxidase (about 4 times) and no changes in lipoperoxidation levels. We suggest that the higher activity of this enzyme induced by ketogenic diet in hippocampus might contribute to protect this structure from neurodegenerative sequelae of convulsive disorders.     

亚硒酸钠对小麦幼苗中谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及谷胱甘肽含量的影响

用1.0 mg·L-1的亚硒酸钠根施小麦幼苗,测定亚硒酸钠对谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及还原性谷胱甘肽含量的结果表明,外源亚硒酸钠对麦苗地上部的谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性均有诱导作用,使麦苗体内的谷胱甘肽含量水平增加.     

Doxorubicin Resistance Conferred by Selective Enhancement of Intracellular Glutathione Peroxidase or Superoxide Dismutase Content in Human MCF-7 Breast Cancer Cells

To examine the role of doxorubicin-stimulated oxyradical formation in tumor cell killing, we introduced glutathione peroxidase (GSH Px) or superoxide dismutase (SOD) into MCF-7 cells by “scrape loading.” Control cytoplasmic GSH Px and SOD levels increased from (mean ± S.E.) 0.37nmol/min/mg and 0.58 μg SOD/mg, respectively, to 3.99 or 7.63 nmol/min/mg and 1.40 or 1.83 μg SOD/mg after treatment with either 150 or 300 units/ml of GSH Px or 20 or 40mg/ml SOD. Resistance to doxorubicin was cbrrelated with the level of GSH Px introduced into the MCF-7 cells: a one-hour exposure to 1.75 μM doxorubicin decreased the cloning efficiency of control cells loaded with medium alone to 34%, whereas doxorubicin-treated cells augmented with 150 or 300 units/ml of GSH Px had plating efficiencies of 56 or 86%, P < 0.05. Introduction of SOD increased MCF-7 resistance to doxorubicin similarly. The heat-inactivated enzymes were not protective. Cells loaded with GSH Px were also resistant to the redox cycling anticancer quinone mitomycin C but not to the redox inactive analogs 5-iminodaunorubicin and mitoxan-trone. suggesting that amplification of GSH Px or SOD levels can produce doxorubicin resistance in MCF-7 cells.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Kupffer Cell Function in Thyroid Hormone-Induced Liver Oxidative Stress in the Rat

The influence of thyroid hormone (L-3, 3', 5-triiodothyronine, T₃) on Kupffer cell function was studied in the isolated perfused rat liver by colloidal carbon infusion. Rates of carbon uptake were determined from the influent minus effluent concentration difference and the flow rate, and the respective carbon-induced respiratory activity was calculated by integration of the area under the O₂ curves during carbon infusion. In the concentration range of 0.2 to 2.0 mg of carbon/ml, livers from euthyroid rats exhibited a sigmoidal-type kinetics of carbon uptake, with a Vmax of 4.8 mg/g liver/min and a concentration of 0.82 mg/ml for half-maximal rate; carbon-induced O₂ uptake presented a hyperbolic-type kinetics, with a Vmax of 4.57 μmol of O₂/g liver and a Km of 0.74 mg of carbon/ml, which significantly correlates with the carbon uptake rates. Light-microscopy showed that carbon was taken up exclusively by non-parenchymal cells, predominantly by Kupffer cells. Thyroid calorigenesis was found in parallel with increased rates of hepatic O₂ consumption and thiobarbituric acid reactive substances (TBARS) formation, glutathione (GSH) depletion, and higher sinusoidal lactate dehydrogenase (LDH) efflux compared to control values. In the concentration range of 0.25 to 0.75 mg/ml, carbon infusion did not modify liver LDH efflux in control rats, while it was significantly enhanced in T₃-treated animals. In this latter group, higher carbon concentrations (1 and 1.3 mg/ml) led to loss of viability of the liver. At 0.25 to 0.75 mg of carbon/ml, both the rates of carbon uptake and the associated carbon-induced respiratory activities were significantly increased by T₃ treatment, effects that were abolished by pretreatment of the rats with gadolinium chloride (GdCl₃). In addition, GdCl₃ decreased by 50% the changes induced by T₃ in hepatic GSH content and TBARS formation. It is concluded that hyperthyroidism enhances Kupffer cell function, correlated with the increased number of liver macrophages observed histologically, which may represent an alternate source of reactive O₂ species to that induced in parenchymal cells, thus contributing to the enhanced oxidative stress status developed.     

Glutathione cycle in stable chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low‐molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (n = 109) and healthy subjects (n = 51). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never‐smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase‐dependent regulation of the glutathione redox state is vital for protection against oxidative stress. Copyright © 2010 John Wiley & Sons, Ltd.     

A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25-2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.