钙对玉米幼苗谷胱甘肽还原酶活性的影响

CaCl2浸种或酶提取液中加入CaCl2均可显著提高两品种玉米幼苗中谷胱甘肽还原酶（GR）活性,且不同钙盐对GR的激活效应基本相同。Ca^2 专一螯合剂EGTA浸种或加入到酶提取液中则可显著抑制GR活性,表明GR活性至少部分受Ca^2 调控。     

谷胱甘肽还原酶和超氧化物歧化酶在玉米幼苗热激诱导的交叉适应中的作用

玉米幼苗经过热激处理后,在随后的高温、低温、干旱和盐胁迫环境中,其存活率明显高于未热激的幼苗,表明热激能提高植物的抗热性、抗冷性、抗旱性和抗盐性,证实了玉米幼苗交叉适应现象的存在;热激还可提高谷胱甘肽还原酶(GR)和超氧化物歧化酶(SOD)的活性,且在上述4种胁迫过程中GR、SOD活性水平与玉米幼苗的存活率呈正相关,表明GR和SOD参与玉米幼苗交叉适应的形成.     

钙对渗透胁迫下玉米幼苗内源激素和多胺含量的影响(简报)

渗透胁迫下,玉米幼苗叶片中内源ABA含量增加,乙烯在较短时间内出现峰值,叶中腐胺（Put）、亚精胺（Spd）和精胺（Spm）先降后升。喷施CaCl2水溶液后叶中ABA增加的起始时间延迟,乙烯释放量减少,Put、Spd、Spm含量均增高。     

亚硝基化谷胱甘肽还原酶: 一个调控炎症反应的新分子

炎症因子的表达调控是炎症反应的关键步骤,与自身免疫疾病以及癌症等密切相关．一氧化氮(nitric oxide,NO)在炎症因子表达调控中具有重要作用,但已有的研究多关注于NO合成对炎症因子的调控作用,而对NO代谢的作用知之甚少．亚硝基化谷胱甘肽还原酶(S-nitrosoglutathione reductase,GSNOR)是体内NO信号通路代谢调控的关键蛋白．本研究发现脂多糖(lipopolysaccharide,LPS)在RAW264.7细胞中上调诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的同时下调GSNOR的转录和蛋白质表达,该下调作用依赖MEK1/2、p38和PI3K信号通路．抑制GSNOR可促进LPS诱导的炎症因子IL-1β、IL-6和TNF-α表达,而过表达GSNOR作用相反．抗炎症药物曲古抑菌素A (trichostatin A,TSA)能够挽回LPS对GSNOR的下调作用,并且GSNOR抑制剂削弱了TSA对炎症因子IL-6和TNF-α转录的抑制效应．这些结果表明：GSNOR是一个新的重要炎症调控分子,它可能成为调控NO介导的炎症相关信号通路的新的潜在靶点,上调GSNOR可能是抑制炎症的新思路．本研究揭示了巨噬细胞通过上调iNOS和下调GSNOR共同增强免疫炎症反应的新机制,拓展了对NO代谢在炎症反应中作用机制的认识．     

钙对玉米幼苗热激诱导抗盐性的影响

热激处理的玉米幼苗抗盐性提高,盐胁迫期间的谷胱甘肽还原酶(GR)活性和脯氨酸含量也较高.Ca2 处理可增强热激诱导的玉米幼苗抗盐性,GR活性和脯氨酸含量也进一步提高;Ca2 螯合剂EGTA处理的效果正好相反.     

逆境胁迫对油菜谷胱甘肽还原酶基因表达及其酶活性的影响

利用cDNA末端快速分离(RACE)技术从陇油6号油菜中克隆得到一个新的谷胱甘肽还原酶基因GR2,全长2073 bp,开放阅读框1692 bp,编码563个氨基酸,预测蛋白质分子量为60.7 kDa,等电点7.9.实时荧光定量PCR分析表明:GR2基因在油菜根、茎、叶中均有表达,其中在叶中表达量最高.GR1和GR2基因的转录以及谷胱甘肽还原酶(GR)活性受到低温、高温、干旱、高盐胁迫的诱导,表明油菜谷胱甘肽还原酶在抵御低温、高温、干旱、高盐胁迫过程中发挥重要作用.脱落酸(ABA)预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显上升,表明ABA可以诱导GR1和GR2基因表达和GR酶活性.MAPKK抑制剂U0126预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显下降,表明U0126对GR1、GR2基因表达以及GR酶活性有抑制作用.     

涝渍逆境下玉米叶片中谷胱甘肽的含量变化及其作用

涝渍逆境下玉米叶片ＧＳＨ含量和ＧＳＨ还原酶活性都明显下降,但前者的降幅小于后者,两者的变化均与叶片质膜透性以及ＭＤＡ的积累呈显著性负相关。施加外源ＧＳＨ明显减少ＭＤＡ和Ｈ２Ｏ２含量,降低Ｏ＾－２产生速率。而叶面喷施８－羟基喹啉和抗坏血酸等活性氧清除剂则减缓因涝渍而引起的ＧＳＨ含量下降。     

SO_2对玉米幼苗POD、Pro和MDA的影响

本文采用室内水培及密闭箱静态熏气方法,研究了4个不同浓度(0mg/m3,10mg/m3,30mg/m3和90mg/m3)SO2对晋单51和农大108两个品种玉米幼苗过氧化物酶(POD)、脯氨酸(Pro)和丙二醛(MDA)的影响。研究结果表明,随着SO2浓度的升高,两种玉米幼苗的POD活性呈下降的趋势,且当SO2浓度为10mg/m3、30mg/m3和90mg/m3时POD活性分别降至对照的22.6%、18.9%和12.1%;而结果发现Pro含量随SO2浓度升高逐渐升高,且当SO2浓度与POD处理相同时脯氨酸含量分别上升至对照的195.0%、235.4%和496.5%,SO2浓度为90mg/m3时Pro含量最高,且不同浓度处理时农大108Pro含量均比晋单51高;然而,MDA含量随SO2浓度的升高呈先降低后逐渐升高的趋势,在90mg/m3处升至最高值为对照的310.6%,仍然是农大108MDA含量比晋单51高。因此研究显示低浓度SO2能使玉米幼苗适应胁迫,但高浓度SO2暴露会影响植物生长发育。     

Ca~(2 )对盐胁迫下黄瓜幼苗中CaM、MDA含量和质膜透性的影响(简报)

添加CaCl2可提高黄瓜幼苗中CaM含量,降低MDA（丙二醛）和质膜透性,减轻盐害,添加CaM抑制剂CPZ（氯丙嗪）、TFP（三氟拉嗪）则取得相反效果。添加外源CaM可降低CaM在体内合成,起到降低盐害作用。     

胞外Ca~(2 )促进粟酒裂殖酵母细胞增殖作用的研究

研究了胞外Ca~(2 )对粟酒裂殖酵母（Schizosaccharomycespombe）细胞增殖的影响。实验结果首次证明胞外Ca~(2 )能明显促进粟酒裂殖酵母的增殖,其作用方式主要是缩短了粟酒裂殖酵母的生长延滞期。当起始的接种细胞密度升高至使粟酒裂殖酵母的生长延滞期消失时,外加Ca~(2 )的作用也消失。EGTA可抑制粟酒裂殖酵母的细胞增殖,而外加Ca~(2 )能够有效消除EGTA的抑制作用,进一步说明胞外Ca~(2 )是粟酒裂殖酵母增殖所必需的。此外,外加EGTA除了可延长细胞增殖的延滞期外,还能显著降低指数期细胞分裂的速率以及达到稳定期时培养液中的细胞总数,提示缺Ca~(2 )还影响粟酒裂殖酵母细胞的分裂。     

Differential Expression of Glutathione Reductase and Cytosolic Glutathione Peroxidase,GPX1, in Developing Rat Lungs and Kidneys

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.     

水分胁迫及复水过程中小麦幼苗叶片内Ca2+的定位

展现了冬小麦幼苗在干旱胁迫及干旱后复水过程中叶肉细胞内Ca2 的动态分布 :在正常水分条件下生长的小麦幼苗 ,其细胞中的Ca2 主要位于液泡内 ,同时 ,细胞间隙中有大量的Ca2 分布。在水分胁迫下 ,随着胁迫时间的加长 ,液泡和细胞间隙的Ca2 逐渐进入细胞质 ,导致细胞质中自由Ca2 浓度过高 ,并对细胞造成伤害。复水后 ,细胞质中高浓度Ca2 迅速排入液泡和细胞间隙 ,细胞质中Ca2 浓度又基本恢复正常水平 ,形象地展示了细胞内Ca2 的稳态调控机制     

Inhibitors of Glutathione Reductase as Potential Antimalarial Drugs Kinetic Cooperativity and Effect of Dimethyl Sulphoxide on Inhibition Kinetics

Abstract

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with K_i and α values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with K_i and α values of 0.4μM and 0.15, respectively. LY83583 and 2-anilino-l,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar K_i values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a K_i value of 11 μM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by pre-incubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.     

Thermal Kinetics of Glutathione Reductase and their Relation to Thermotolerance in Diverse Cultivars of Maize

The characteristics of glutathione reductase from a number ofmaize cultivars with contrasting thermotolerance have been investigated.The apparent K_m (Michaelis constant) for oxidised glutathione(GSSG) was measured between 10 and 45°C at constant pH.The enzyme from highland cultivars adapted to cool environmentshad a slightly lower apparent K_m for GSSG than that from lowlandtropical cultivars at low assay temperatures. Similarly theenzyme from lowland tropical cultivars had a lower apparentK_m for GSSG at high assay temperatures. However these effectswere small and regression lines plotted through the data werenot significantly different in slope or intercept. There wasa strong correlation (r = 0·939) between apparent K_mand V_max (maximum initial velocity) as assay temperature wasvaried. The interpretation of apparent K_m/temperature relationshipsis discussed with hypothetical examples of the effects of temperatureon enzyme activity/substrate concentration plots. It is demonstratedthat an increase in apparent K_m at higher assay temperaturesneed not necessarily reflect any temperature-dependent impairmentof enzyme function. The apparent K_m for GSSG of glutathionereductase from maize increased over four-fold when the temperaturewas raised from 10 to 45°C, but it is concluded that invivo rates of reaction are likely to be increased rather thandecreased by this same change in temperature. Glutathione reductasewould thus appear to be equally well adapted to function atall these temperatures. This suggests that the potential ofenzyme thermal kinetics to predict thermotolerance may be limited.Copyright1994, 1999 Academic Press Zea mays L., maize, glutathione reductase, thermal kinetics, thermotolerance     

Mitochondrial Glutathione Transport Is a Key Determinant of Neuronal Susceptibility to Oxidative and Nitrosative Stress

Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.     

Induction of Nitrate Reductase and Peroxidase Activity in the Seedlings of Normal and High Lysine Maize

Steady state levels of in vivo nitrate reductase activity in the endosperm, scutella, roots and shoots of maize seedlings were higher in normal maize than those in high lysine maize. Activity of peroxidase in the roots, however, was higher in the high lysine cultivar. The nitrate reductase activity increased with the supply of nitrate in all parts of the seedlings of both cultivars, although the rates of increment in the endosperm were lower than those in scutella, roots and shoots. In relation to substrate concentration, a saturation was achieved at 5 to 10 mM of nitrate except in the endosperm, where activity increased slowly up to 100 mM at least. Final levels of enzyme activity were significantly higher in the scutella of normal than in that of high lysine seedlings. In vitro enzyme activity in the roots also increased with the supply of nitrate in both cultivars, reaching maximum at 5 to 10 mM nitrate.     

Identification of the Binding Site of Methylglyoxal on Glutathione Peroxidase: Methylglyoxal Inhibits Glutathione Peroxidase Activity via Binding to Glutathione Binding Sites Arg 184 and 185

Methylglyoxal (MG), a physiological &#102 -dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272 , 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.