磷脂化修饰影响超氧化物歧化酶细胞亲和力的研究

目的:研究磷脂化修饰对重组人超氧化物歧化酶(SOD)进入人冠状动脉内皮细胞(HCAEC)、人心肌细胞(HCM)能力的影响。方法:分别运用流式细胞术和蛋白印迹分析磷脂化修饰的超氧化物歧化酶(PC-SOD)和SOD与HCAEC、HCM的结合能力,并用激光共聚焦显微术分析修饰前后的SOD可显著增强PC-SOD与细胞的亲和力,并可显著增强PC-SOD进入人冠状动脉内皮细胞和人心肌细胞的能力。     

小胸鳖甲胞外铜锌超氧化物歧化酶重组蛋白的原核表达及多克隆抗体制备

超氧化物歧化酶(SOD)是清除生物体内超氧阴离子自由基的主要抗氧化酶家族。基于原核表达系统,成功表达了拟步甲科Tenebrionidae小胸鳖甲Micordera punctipennis胞外铜锌SOD的重组蛋白(本文定义为Trx-His-MpecCu/Zn-SOD)。经Ni 2+亲和层析法纯化重组蛋白后,研究了重组蛋白的部分酶学性质。通过足垫加皮下注射法3次免疫小鼠后,分别用ELISA和Western blot的方法检测抗体效价和抗体特异性。结果表明,重组蛋白主要以包涵体形式存在,纯化后的重组蛋白浓度为1.33 mg·mL^-1,酶活力为27.52 U·mg^-1。Trx-His-MpecCu/Zn-SOD在25~45℃具有比较稳定的酶活性,在35℃最高,同时表现出比较广泛的酸碱耐受性(pH3~12),最适pH为9.0,表明重组蛋白的酶活性相对比较稳定。蛋白免疫法制备的鼠抗MpecCu/Zn-SOD多克隆抗体滴度高于1∶819 200。Western blot结果显示,该抗体能免疫结合重组蛋白Trx-His-MpecCu/Zn-SOD和小胸鳖甲体内天然MpecCu/Zn-SOD,但不能与黄粉虫Tenebrio molitor的总蛋白结合,说明制备的抗体效价较高且特异性较好。本研究结果为小胸鳖甲ecCu/Zn-SOD功能的深入研究奠定了基础。     

Age-Dependent Resistance to the Toxic Effects of Paraquat in Relation to Superoxide Dismutase Activity in Rat Lung

Young rats are much more resistant to the toxic effects of orally-administered paraquat than are older rats. The possible relation of this phenomenon to the activity of superoxide dismutase in lung is discussed.     

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。     

Purification, N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

Catalase and Superoxide Dismutase in the Cells of Strictly Anaerobic Microorganisms

Strictly anaerobic microorganisms relating to various physiological groups were screened for catalase and superoxide dismutase (SOD) activity. All of the investigated anaerobes possessed SOD activity, necessary for protection against toxic products of oxygen reduction. High specific activities of SOD were found in Acetobacterium woodii and Acetobacterium wieringae. Most of the investigated clostridia and acetogens were catalase-negative. A significant activity of catalase was found in Thermohydrogenium kirishiense, in representatives of the genus Desulfotomaculum, and in several methanogens. Methanobrevibacter arboriphilus had an exceptionally high catalase activity after growth in medium supplemented with hemin. Hemin also produced a strong positive effect on the catalase activity in many other anaerobic microorganisms. In methanogens, the activities of the enzymes of antioxidant defense varied in wide ranges depending on the stage of growth and the energy source.     

Purification,N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

橄榄超氧化物歧化酶的分离纯化与性质研究

采用超声波破碎、硫酸铵分级沉淀、SephadexG-100和DE-52柱层析,从橄榄中分离纯化Cu,Zn-SOD,并对其部分性质进行分析鉴定。结果得到酶的比活力为906.3U/mg。该酶对H2O2和KCN敏感,而T氏液对酶活性没影响。紫外吸收峰在275nm处,PAGE蛋白和活性染色呈现3条相对应的谱带,相对分子质量约为31.63kD,亚基相对分子质量约为15.73kD。此酶对热稳定,在pH7~9范围内稳定。     

蚯蚓超氧化物歧化酶理化性质的研究

从蚯蚓中提取超氧化物歧化酶(Superoxide Dismutase简称SOD),经硫酸铵分级沉淀分离、透析除盐后测定SOD活性得知:50~70%饱和度硫酸铵沉淀的SOD活性最高,比活性也最高;蚯蚓体内此酶在35~40℃之间表现出最高活性;最适pH值为7.8~8.2;提取蚯蚓SOD的最适提取液体积应为蚯蚓鲜重的4倍(v/w)。     

Pro-Oxidant Action of Superoxide Dismutase in the Autoxidation of Rifamycin Sv

6-ketocholestanol. a naturally occurring oxygenated sterol, when incubated with human neutrophils (PMN), can inhibit superoxide and hydrogen peroxide generation in a dose-dependent fashion. This is accompanied by inhibition of stimulated PMN aggregation without alteration in cellular viability. This inhibitory effect is not affected by washing of the cells, and cannot be blocked by the addition of free cholesterol to the medium. These data are consistent with prior observations which showed an inhibitory effect on PMN chemotaxis by certain oxygenated sterol compounds. and support the hypothesis that certain oxygenated sterols can affect a variety of human PMN functions by a mechanism that may involve perturbation of the plasma membrane.     

In vitro Damage to Rat Lens by Lumazine and Xanthine Oxidase: Prevention by Superoxide Dismutase

Intact rat lenses incubated with lumazine and xanthine oxidase are physiologically damaged as evidenced by a decrease in the net accumulation of rubidium ions against a concentration gradient. Superoxide dismutase protected the tissue against this damage. These experiments, therefore, demonstrate the susceptibility of the lens tissue to O₂^?_? injury under ambient and nonphotochemical conditions, suggesting a possible implication of this radical in the tissue in vivo and eventual cataract formation. The lumazine/xanthine oxidase system which is known to cause oxygen reduction predominantly by the monovalent route, producing superoxide, appears quite suitable to evaluate the toxicity of O₂^?_? to the tissues in vitro.     

超氧化物歧化酶在反胶束体系中稳定性的研究

目的：考察反胶束体系组成对超氧化物歧化酶(SOD)抗氧化活性的影响。方法：以大豆磷脂氧化产生的氧自由基量为检测指标,考察温度、有机试剂以及表面活性剂浓度对反胶束体系中超氧化物歧化酶抗氧化活性的影响。结果：60℃时,反胶束中超氧化物歧化酶的抑制率由在水溶液中的52．48%升到75．38%,即SOD包封于反胶束中后热稳定性明显提高。表面活性剂可有效阻止有机溶剂对SOD的变性作用,SOD是不具界面活性的酶。结论：超氧化物歧化酶在反胶束体系中稳定性及抗氧化活性增强。     

人锰超氧化物歧化酶基因剪接异构体的表达分析

对人锰超氧化物歧化酶(human manganese superoxide dismutase,hMn-SOD)基因剪接异构体进行分析,并检测异构体的表达情况。在GenBank库中检索人锰超氧化物歧化酶基因异构体及编码基因组序列,利用Vector NTI9生物软件进行核酸及蛋白序列比对;利用RT-PCR方法分析锰超氧化物歧化酶基因异构体的表达。结果显示,在GenBank库检索发现有3种人锰超氧化物歧化酶基因异构体,剪接异构的类型为可变的5′剪接位点和外显子盒,各异构体基因内含子均符合"GT-AG"规则。3种基因异构体编码两种异构体蛋白,即222个氨基酸的人锰超氧化物歧化酶蛋白以及中部缺少39个氨基酸的截短型异构蛋白。RT-PCR检测结果表明,剪接异构体hMn-SODb在HEK293T和HSC细胞中的表达比在HepG2细胞中高,未见异构体hMn-SODc的表达。     

四棱豆不同节位各器官中SOD活性的测定

测定实验表明 :位于四棱豆植株中部节位叶片中的SOD活性高于植株下部和上部节位叶片中的SOD活性 ;而位于下部节位豆荚和种子中的SOD活性要高于位于上部节位豆荚和种子中的SOD活性 ,且随着结荚部位升高 ,SOD活性呈降低趋势。     

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobocterium leiognothi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide disrnutases shows 21 invariant residues in key positions related to maintenance of the β-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobocterium Cu/Zn superoxide dismutase. Copper/zinc superoxide disrnutase from the trematode Schisiosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide. as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase. including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of β-barrel fold and active site structure for the extracellular enzyme.