小立碗藓GPX家族的功能分化与催化特性研究

谷胱甘肽过氧化物酶(GPX)在植物抵抗氧化胁迫中发挥重要作用。该研究从小立碗藓(Physcomitrella patens)基因组中挖掘到3个GPX基因,分别命名为PpGPX1、PpGPX2和PpGPX3。其中PpGPX1和PpGPX3只含有1个外显子,而PpGPX2含有6个外显子。表达模式分析发现PpGPX1和PpGPX2在检测的所有条件下均表达,而PpGPX3在检测的所有条件下均不表达。蛋白亚细胞定位分析发现,PpGPX1蛋白定位在细胞质,而PpGPX2蛋白定位在叶绿体。在大肠杆菌中表达并纯化了PpGPX1和PpGPX2蛋白,酶学性质分析发现,PpGPX1和PpGPX2蛋白均只能利用Trx电子供体系统,而不能利用GSH电子供体系统;PpGPX2蛋白对过氧化物底物的催化活性和催化效率均高于PpGPX1。基因结构、表达模式、亚细胞定位和蛋白酶学性质的差异预示小立碗藓GPX基因家族成员发生了功能分化,将PpGPX2蛋白的Pro158、Phe167和Phe172氨基酸残基均突变为Ala,发现突变体蛋白对底物催化活性降低,说明这3个氨基酸位点对PpGPX2蛋白具有重要催化活性。     

Glutathione Peroxidase from Mucor hiemalis

Asymmetric epoxidation of cyclic enones was performed with 9-alkylfluorenyl peroxides under two-phase conditions in the presence of novel phase transfer catalysts derived from cinchona alkaloids. The observed enantiomeric excess ranged between 30~63%, from which it is shown that the fluorenyl group had a remarkable effect on the enhancement of enantioselectivity.     

油松雌性不育系球果蛋白质双向电泳技术的建立

本文建立了油松雌性不育系雌球果蛋白质组研究中的双向电泳技术。第一向采用固定pH值梯度（IPG）胶条在IPGphorTM等电聚焦仪上进行等电聚焦,第二向在恒功率且恒温条件下于Ettan-DALTTMⅡ高通量电泳仪上进行SDS-PAGE电泳,以银染和考马斯亮蓝两种方法染色。通过对全蛋白的提取、胶条pH值和胶条肿胀等技术环节的优化和比较,得到了重复性很高,分离效果良好的蛋白质双向图谱。     

油松雌性不育系球果蛋白质双向电泳技术的建立

本文建立了油松雌性不育系雌球果蛋白质组研究中的双向电泳技术.第一向采用固定pH值梯度(IPG)胶条在IPGphorTM等电聚焦仪上进行等电聚焦,第二向在恒功率且恒温条件下于Ettan-DALTTMⅡ高通量电泳仪上进行SDS-PAGE电泳,以银染和考马斯亮蓝两种方法染色.通过对全蛋白的提取、胶条pH值和胶条肿胀等技术环节的优化和比较,得到了重复性很高,分离效果良好的蛋白质双向图谱.     

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.     

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.     

人肝谷胱甘肽过氧化物酶纯化及性质研究

经硫酸铵分级沉淀,离子交换层析和凝胶过滤等步骤,从人肝中获得了ＰＡＧＥ单一条带的谷胱甘肽过氧化物酶,比活提高１２０倍,得率为２５％。凝胶过滤法测得分子量为９０９８０,ＳＤＳ－ＰＡＧＥ测定亚基分子量为２２４２３．原子吸收法测得每分子酶含有四个硒原子。等电聚焦显示该酶等电点为５．０．酶活力的最适ｐＨ为８．５,最适温度为３７℃。动力学实验提示该酶作用机理属于乒乓机制型。     

Catalytic properties of glutathione-binding residues in a tau class glutathione transferase (PtGSTU1) from Pinus tabulaeformis

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification in plants. However, there is extremely little information on the molecular characteristics of GSTs in gymnosperms. In a previous study, we cloned a tau class GST (PtGSTU1) from a gymnosperm (Pinus tabulaeformis) for the first time. Based on the N-terminal amino acid sequence identity to the available crystal structures of plant tau GSTs, Ser13, Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 were proposed as glutathione-binding (G-site) residues. The importance of Ser13 as a G-site residue was investigated previously. The functions of Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 are examined in this study through site-directed mutagenesis. Enzyme assays and thermal stability measurements on the purified recombinant PtGSTU1 showed that substitution at each of these sites significantly affects the enzyme's substrate specificity and affinity for GSH, and these residues are essential for maintaining the stability of PtGSTU1. The results of protein expression and refolding analyses suggest that Ile54 is involved in the protein folding process. The findings demonstrate that the aforementioned residues are critical components of active sites that contribute to the enzyme's catalytic activity and structural stability.     

油松菌根根际放线菌的分离与鉴定

为了探讨油松菌根根际放线菌与菌根真菌及油松猝倒病的关系,该实验从油松菌根根际土中分离、纯化得到170株放线菌,采用平板对峙培养法对油松猝倒病病原真菌立枯丝核菌(Rhizoctonia solani)和茄腐皮镰孢霉(Fusarium solani)进行生防放线菌的初筛,得到7株对病原真菌抑制效果较好的放线菌.用初筛选的7株放线菌菌株分别与外生菌根真菌灰鹅膏菌(Amanita vatinata)、灰环粘盖牛肝菌(Suillus laricinus)和血红铆钉菇(Gomphidius viscidus)进行纯培养互作复筛,得到2株放线菌(菌株19#、菌株110#)对外生菌根真菌灰鹅膏菌和血红铆钉菇均具有促生作用.将菌根真菌灰鹅膏菌、放线菌菌株19#、病原真菌立枯丝核菌进行纯培养互作试验表明,菌株19#能协同灰鹅膏菌拮抗立枯丝核菌.经形态特征、培养特征、生理生化特性测定和16SrDNA序列分析,确定菌株19#为苯胺紫链霉菌(Streptomyces mauvecolor).     

Inactivation of Glutathione Peroxidase by Peroxynitrite

Glutathione peroxidase (GSH-Px) is inactivated on exposure to peroxynitrite under physiologically relevant conditions. Stopped-flow kinetic studies show that the reaction between peroxynitrite and GSH-Px is first-order in each of the reactants, with an apparent second-order rate constant of 4.5 ± 0.2 × 10⁴M⁻¹s⁻¹per monomer unit of enzyme. In good agreement with this value, GSH-Px inactivation experiments afford an apparent second-order rate constant of 1.8 ± 0.1 × 10⁴M⁻¹s⁻¹per monomer unit of enzyme. The hydroxyl radical scavengers mannitol, DMSO, and benzoate (at 100 mM) afford only 8–12% protection of the enzyme, while addition of 25 mM bicarbonate results in 55% protection. The minimal protection by hydroxyl radical scavengers indicates, as expected, that hydroxyl radicals are not involved in the inactivation. Protection by bicarbonate occurs because peroxynitrite is rapidly trapped by CO₂to form the adduct nitrosoperoxycarbonate (ONOOCO⁻₂), and/or other reactive species that preferentially decompose to nitrate rather than react with GSH-Px. The close agreement between the rate constants obtained from enzyme inactivation and from stopped-flow kinetics experiments suggests that the mechanism of the reaction between peroxynitrite and GSH-Px involves the oxidation of the ionized selenol of the selenocysteine residue in the enzyme's active site (E-Se⁻) by peroxynitrite. This reaction does not simply involve formation of the selenenic acid, E-SeOH, because E-SeOH is an intermediate in the catalytic cycle of the enzyme, and thus its formation cannot explain the inactivation we observe. Thus, the ionized selenol in the active site is transformed into a form of selenium that cannot easily be reduced back to the selenol.     

兔肝谷胱甘肽过氧化物酶的亲和层析纯化

<正> 谷胱甘肽过氧化物酶(GSH-PX,EC1.11.1.9)与过氧化氢酶、超氧化物歧化酶一起共同构成细胞内抗脂质过氧化作用的酶性保护系统。近年来,随着人们对脂质过氧化作用机理及其对细胞组织损伤和遗传物质变异作用的认识的日趋深入,GSH-PX越来越受到人们的重视。关于GSH-PX的提纯已有大量的文献报导,但利用共价亲和层析来分离提纯此酶的文献报导则较少。本文根据GSH-PX的催化机理及其存在的不同状态,发展了一种独特的共价亲和层析的提纯方法。     

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.     

The Physiological Properties of a Novel Family of VDAC-Like Proteins from Drosophila melanogaster

VDAC, a major protein of the mitochondrial outer membrane, forms voltage-dependent, anion-selective channels permeable to most metabolites. Although multiple isoforms of VDAC have been found in different organisms, only one isoform (porin/DVDAC) has been previously reported for Drosophila melanogaster. We have examined the physiological properties of three other Drosophila proteins (CG17137, CG17139, and CG17140) whose primary sequences have significant homology to DVDAC. A comparison of their hydropathy profiles (β-pattern) with known VDAC sequences indicates the same fundamental folding pattern but with major insertions and deletions. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137 and CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 was not able to form channels. DVDAC and CG17137 were able to rescue the temperature-sensitive conditional-lethal phenotype of VDAC-deficient yeast, whereas CG17139 and CG17140 demonstrated no complementation. Similar structure and channel formation indicate that VDAC-like proteins are part of the larger VDAC family but the modifications are indicative of specialized functions.     

谷胱甘肽过氧化物酶的硒代半胱氨酸插入元件

真核生物将硒代半胱氨酸插入蛋白质必需硒代半胱氨酸插入元件（ＳＥＣＩＳ）的参与,后者位于硒蛋白ｍＲＮＡ的３′非翻译区．采用ＲＮＡ折叠程序对１５个谷胱甘肽过氧化物酶基因进行计算机处理发现,其可能的ＳＥＣＩＳ中都具有３段保守碱基ＡＵＧＡ－Ａ（Ｇ）ＡＡ－ＧＡ．根据Ａ（Ｇ）ＡＡ位于顶环或者顶环上游５′臂的突环上,可将ＳＥＣＩＳ分为Ⅰ型和Ⅱ型结构     

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.     

黄龙山油松林和油松+辽东栎混交林物种组成及优势种群动态

根据黄龙山油松(Pinus tabulaeformis)林和油松+辽东栎(Quercus liaotungensis)混交林共6个群丛中27个典型样地调查资料,分析了松栎林群落物种组成、多样性指数和优势种群动态.结果表明:(1)松栎混交林中灌木层和草本层植物种数均大于油松林,且灌木和草本种类因群落生境不同而有差异.(2)物种多样性分析显示,油松林和松栎混交林中由于生境不同导致灌木层物种多样性差异最大.(3)优势种群分析表明,油松林中阴坡幼苗数量较多,种群最大的为Ⅳ和Ⅴ龄级个体;松栎混交林中油松和辽东栎幼苗充足,到Ⅱ、Ⅲ龄级二者均大量减少,此后油松个体增加而辽东栎减少,说明松栎林发育过程中各层次植物物种相互制约,优势种群之间也存在竞争,且受生境条件影响较大.(4)主成分分析表明,气温、土壤、光照和总郁闭度是主要影响因子.因此,在未来森林抚育中应适当减少优势种群密度,促进异龄化;采取有效措施合理搭配灌木和草本,强化对森林发育有利的因素,提高林分质量.     

Characterization of Dye-decolorizing Peroxidase (DyP) from Thermomonospora curvata Reveals Unique Catalytic Properties of A-type DyPs

Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which has received much attention due to their potential applications in lignin degradation. A new DyP from Thermomonospora curvata (TcDyP) was identified and characterized. Unlike other A-type enzymes, TcDyP is highly active toward a wide range of substrates including model lignin compounds, in which the catalytic efficiency with ABTS (k_cat^app/K_m^app = (1.7 × 10⁷) m⁻¹ s⁻¹) is close to that of fungal DyPs. Stopped-flow spectroscopy was employed to elucidate the transient intermediates as well as the catalytic cycle involving wild-type (wt) and mutant TcDyPs. Although residues Asp²²⁰ and Arg³²⁷ are found necessary for compound I formation, His³¹² is proposed to play roles in compound II reduction. Transient kinetics of hydroquinone (HQ) oxidation by wt-TcDyP showed that conversion of the compound II to resting state is a rate-limiting step, which will explain the contradictory observation made with the aspartate mutants of A-type DyPs. Moreover, replacement of His³¹² and Arg³²⁷ has significant effects on the oligomerization and redox potential (E°′) of the enzyme. Both mutants were found to promote the formation of dimeric state and to shift E°′ to a more negative potential. Not only do these results reveal the unique catalytic property of the A-type DyPs, but they will also facilitate the development of these enzymes as lignin degraders.     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.