羊血铜锌超氧化物歧化酶(Cu-ZnSOD)的分离纯化

目的:对羊血进行提取SOD。方法:采用有机溶剂去除血红蛋白、热变性、丙酮沉淀、DEAE-32离子交换柱层析的方法,对羊红细胞Cu,Zn-SOD进行分离纯化。利用非变性凝胶电泳NBT活性染色鉴定SOD,SDS-PAGE测定分子量。结果:表明1 000ml羊血中得到SOD干粉1 257mg,总活力为79 453U,比活力为3 871.4U/mg。活性染色结果证明羊血SOD有2条带,表明已达到电泳纯。SDS-PAGE测得SOD亚基分子量分别为16.71kDa、15.97kDa。H2O2对羊血CuZn-SOD活性有抑制作用,通过紫外光谱扫描,羊血SOD在231nm处有最大吸收峰。     

脲对果菠萝蛋白酶活力与构象的影响

本文用荧光光谱,紫外差示光谱和CD谱研究果菠萝蛋白酶在不同浓度的脲溶液中的构象及酶活力的变化情况。酶的荧光强度随脲浓度增大而明显增加,8mol/L脲使荧光强度增强65％,发射峰出现红移。差示谱表明在232nm和288nm出现二个正峰,它们均随脲浓度增大而加剧,前者与主链构象变化有关,而后者与生色基团(Trp、Tyr)的微环境变化相关。CD谱表明:天然酶在208nm和225nm处有二个负峰,脲变性后,225nm的负峰基本上不随脲浓度增大而变化,但208nm峰则明显发生变化并逐渐出现红移,6mol/L以上此峰则完全消失。     

黄瓜Cu/Zn-SOD基因克隆及可溶性表达和纯化

[目的]克隆黄瓜Cu/Zn-SOD基因,并利用大肠杆菌进行可溶性表达、纯化及活性测定。[方法]采用Trizol法提取黄瓜表皮的总RNA,然后设计特异性引物,通过RT-PCR技术克隆获得黄瓜Cu/Zn-SOD基因。该基因与p GEX2T载体相连后,转化大肠杆菌BL21(DE3),摸索可溶性表达方法。利用GST亲和层析方法纯化目标蛋白,SOD酶活性测定采用邻苯三酚法。[结果]成功地克隆了全长为459 bp的黄瓜Cu/Zn-SOD基因,编码152个氨基酸。Protein Blast分析表明其结构域中分别包含Cu2+、Zn2+结合位点,为典型的Cu/Zn-SOD酶。目标蛋白可溶性表达条件是16℃、0. 1 mmol/L IPTG诱导20 h。SDS-PAGE分析表明GST亲和层析成功地获得重组黄瓜Cu/Zn-SOD。活性测定表明两次纯化的蛋白样品SOD酶活力分别为2 328. 9 U/m L、2 144. 7 U/m L。[结论]从黄瓜表皮中克隆获得Cu/Zn-SOD基因,该基因在大肠杆菌系统中获得可溶性表达,纯化后的重组蛋白具有较高的SOD酶活力。     

少孢根霉RT-3胞外产超氧化物歧化酶的研究

利用少抱根霉RT－3行固态发酵,从培养物中可以直接拍提得到SOD。固体发酵培养物发酵前后的酶活分析表明少抱根霉RT－3可向胞外分泌SOD。酶抽提液中含有Cu、Zn型和Mh型SOD。对其初步纯化所得粗酶制剂比活力为444．8u／mg。酶的紫外吸收峰在258nnl。酶在60℃以下较稳定,活性稳定pH为5．5～9．5。     

少孢根霉RT—3胞外产超氧化物歧化酶的研究

利用少抱根霉RT－3行固态发酵,从培养物中可以直接拍提得到SOD。固体发酵培养物发酵前后的酶活分析表明少抱根霉RT－3可向胞外分泌SOD。酶抽提液中含有Cu、Zn型和Mh型SOD。对其初步纯化所得粗酶制剂比活力为444．8u／mg。酶的紫外吸收峰在258nnl。酶在60℃以下较稳定,活性稳定pH为5．5～9．5。     

韭菜细胞溶质超氧化物歧化酶的纯化和性质

经硫酸按沉淀、SephadexG－200凝胶过滤和DEAE－Sephacel层析3个步骤,将韭菜细胞溶质SOD纯化到均一程度。从500g叶片中得到2·4mm酶,酶比活力达13000U／mm蛋白。鉴定该酶是Cu·Zn—SOD。测得酶分子量约为30900道尔顿,亚基分子量约为15900道尔顿,N一末端氨基酸为丙氨酸。该酶在紫外与可见光区吸收峰分别在260urn和680urn。实验表明该酶热稳定性良好。     

重组人超氧化物歧化酶化学修饰的初步研究

在高效表达重组人铜锌超氧化物歧化酶(rh Cu/Zn SOD),并纯化得到比活大于4000单位的 rh Cu/Zn SOD 纯品的基础上,采用活化酯法将聚乙二醇(PEG)与 rhCu/Zn SOD 交联,获得分子量约6万的 PEG-SOD 交联物.经 PEG 修饰的酶稳定性增强,表现为对酸、碱和热的耐受力均较未交联酶高.修饰酶的生物半衰期为15h,是天然酶的90倍,酶活性保留80%以上.还实验观察了修饰剂用量与修饰酶保留活性之间的关系.     

镉、铜和锌胁迫下黑藻活性氧的产生及抗氧化酶活性的变化研究

研究了不同Cd、Cu、Zn处理浓度对黑藻体内活性氧()产生及对抗氧化酶(SOD、POD、CAT)活性的分子毒理学效应以探讨高等水生植物抗氧化酶对重金属胁迫的反应。结果表明,三种重金属都不同程度地加快了产生速率;Cu使SOD、POD、CAT活性下降;Cd也都减弱了SOD和POD活性,而CAT活性在0.5—5mg/L处理浓度时增加;Zn对SOD活性也为抑制作用,当浓度为0.5—5mg/L时POD和CAT活性都上升。关联度分析发现Cd、Cu和Zn胁迫下黑藻起主要保护作用的分别为SOD、POD和CAT,而SOD最易受到影响。Cd、Cu处理下的叶绿素含量也都呈下降趋势,而0.5—5mg/L的Zn浓度刺激了叶绿素合成。所有Zn处理、0.5mg/L的Cu处理和0.5—1mg/L的Cd处理的叶绿素a/b值都大于对照值。除了Cu使可溶性蛋白含量减少外,0.5—5mg/L的Zn和0.5—1mg/L的Cd都使其含量增加。综合起来,Cu的毒性最强,其次为Cd,Zn最弱。致死阈浓度分别为:Cu:0.5—1mg/L;Cd:1—2mg/L;Zn:5—6mg/L。SOD是评价重金属对沉水植物毒性效应的灵敏指标。黑藻对水环境Cu污染反应敏感。       

Ca~（2＋）对叶绿素a／b钙结合蛋白波谱学特性的影响（Ⅱ）

用钙螯合亲和层析分离纯化得到的叶绿素ａ／ｂ钙结合蛋白,其荧光发射峰在６８０±１ｎｍ,钙结合后导致荧光发射峰强度降低。当再加入ＥＧＴＡ,螯合钙后,其荧光发射峰强度得以部分恢复。该蛋白在结合钙后,其圆二色谱也发生变化。这些结果表明该蛋白质的构象在结合钙后发生了变化。该蛋白质的荧光激发光谱在４４０ｎｍ和４７０ｎｍ处的激发峰表明此蛋白结合有叶绿素ａ和叶绿索ｂ。本文对叶绿素ａ／ｂ钙结合蛋白在光系统Ⅱ中的可能功能进行了讨论。     

牛脑海马NGF分离纯化及微量元素含量检测

将牛脑海马粗提物经盐析、透析、CM-离子交换层析分离纯化得到2个洗脱峰,其中CM2峰含有NGF。利用Western Blot检测结果表明,NGF的分子量约为55 KD和70 KD;PC12细胞法检测其生物学活性,在CM2分离组分作用下,PC12细胞轴突生长效果优于2 ng/mL标准品NGF;原子吸收分光光度法检测脑海马结构微量元素含量,Zn与Cu无差别,Fe与Zn、Cu有差异(P<0.01);CM2组分中Fe与Cu含量无差异,Zn远高于Fe、Cu含量(P<0.01)。     

Effects of cadmium on structure and enzymatic activity of Cu,Zn-SOD and oxidative status in neural cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder disease. Ten percent of the ALS patients are congenital (familial ALS), and the other 90% are sporadic ALS (SALS). It has been shown that mutations found in the Cu,Zn-SOD cause 20% of the familial ALS due to its low enzyme activity. We hypothesized that heavy metals may interfere the structure of Cu,Zn-SOD protein to suppress its activity in some of the SALS. In this study, we expressed and characterized the recombinant human Cu,Zn-SOD under various concentrations of Cu(2+), Zn(2+), and Cd(2+). By atomic absorption spectrophotometry, we demonstrated that adding of cadmium significantly increased the content of cadmium ion, but reduced its Zn(2+) content and enzyme activity of the Cu,Zn-SOD protein. The data of circular dichroism spectra demonstrated that the secondary structure of Cu,Zn-SOD/Cd is different from Cu,Zn-SOD, but close to apo-SOD. In addition to the effect of cadmium on Cu,Zn-SOD, cadmium was also shown to induce neural cell apoptosis. To further investigate the mechanism of neural cell apoptosis induced by cadmium, we used proteomics to analyze the altered protein expressions in neural cells treated with cadmium. The altered proteins include cellular structural proteins, stress-related and chaperone proteins, proteins involved in reactive oxygen species (ROS), enzyme proteins, and proteins that mediated cell death and survival signaling. Taken together, in this paper, we demonstrate that cadmium decreases the content of Zn(2+), changes the conformation of Cu,Zn-SOD protein to decrease its enzyme activity, and causes oxidative stress-induced neural cell apoptosis.     

Increased nitration and diminished activity of copper/zinc superoxide dismutase in placentas from diabetic rats

Abstract

Nitration-induced protein damage in the placenta leads to impaired blood flow and deficient feto–placental exchange in diabetic pregnancies. This work studied the effect of nitric oxide and peroxynitrite on Cu/Zn SOD activity in rat placentas and evaluated whether Cu/Zn SOD is nitrated in the placenta from diabetic rats at mid-gestation. Protein nitration was evaluated by EIA, Cu/Zn SOD activity by inhibition of the epinephrine auto-oxidation, Cu/Zn SOD expression by western blot and specific nitration by immunoprecipitation. This study found higher levels of protein nitration (p < 0.001), diminished Cu/Zn SOD activity and enhanced protein expression (p < 0.01) in placentas from diabetic rats. Placental Cu/Zn SOD activity was inhibited by peroxynitrite (p < 0.01). Besides, nitration of Cu/Zn SOD was elevated in placentas from diabetic rats (p < 0.01). These results show that rat Cu/Zn SOD can be nitrated, a modification that could lead to the depressed activity of this enzyme found in placentas from diabetic rats.     

长半衰期重组人超氧化物歧化酶的研制及性质

一种双亲有机化合物聚苯乙烯马来酸丁酯(SMA)经酰胺键与重组人铜锌超氧化物歧化酶(rhCu/Zn SOD)共价交联,制得修饰酶.当42%游离氨基被修饰时,保留酶活力为88%.酶蛋白主链结构在修饰前后变化不大.与天然酶相比,修饰酶的生物半衰期延长了22倍,抗蛋白水解酶能力亦有所增强.     

A Novel Superoxide Dismutase Gene Encoding Membrane-bound and Extracellular Isoforms by Alternative Splicing in Caenorhabditis elegans

We have identified a novel Cu/Zn superoxide dismutase gene (termedSOD-4) in Caenorhabditis elegans. Characterization of its complementaryDNA revealed that the gene encodes two isoforms by alternativesplicing, SOD4-1 and SOD4-2 which differ in their C-terminalexons. Their predicted amino acid sequences include a consensussignal peptide at their N-termini and are homologous to theextracellular-types of Cu/Zn superoxide dismutase in mammals.In addition, SOD4-2 possesses a putative transmembrane domainat the C-terminal region. When transiently expressed in Chinesehamster ovary cells, both types were found in the membranesand SOD4-1 also in the culture fluid. It is, therefore, indicatedthat SOD4-1 is an extracellular form and SOD4-2 a membrane-boundform, the latter representing a novel type of SOD. In C. elegans,SOD4-2 mRNA was found to be preferentially expressed in eggs.     

cDNA cloning,high-level expression,purification, and characterization of an avian Cu,Zn superoxide dismutase from Peking duck

As a special species of avian, Peking duck is often used as a model for exploring effective factors against cardio-cerebrovascular diseases, and therefore investigations of antioxidant enzymes including superoxide dismutase are intriguing. By using 3(')-RACE with a gene-specific primer, a cDNA encoding duck Cu,Zn SOD was amplified from the total RNA extracted from Peking duck liver. Three free cysteine residues are found in the deduced amino acid sequence of duck SOD, among which Cys153 at the carbonyl-terminal is a distinctive feature. Production with a high yield of recombinant duck Cu,Zn SOD was achieved in Escherichia coli after the reconstituted expression vector pET-3a-dSOD was transformed into the bacterial strain BL21(DE3)pLysS. After two steps of anion exchange chromatography, a great quantity of the purified enzyme (100mg/L fermented culture) with an enzymatic activity comparable to that of native duck and bovine SOD was finally obtained. Duck SOD is a homodimer with 153 residues for each subunit. The molecular mass of the recombinant enzyme is 15,540.0Da measured by mass spectrum, which well coincides with the estimated size of the sequence but significantly differs from that of the native counterpart. Five charge isomers were observed on isoelectricfocusing (IEF). The most interesting observation is that the thermal stability of duck SOD is much lower than that of the bovine enzyme as revealed by irreversible heat inactivation at 70 degrees C. These properties are discussed in relation to the distinctive free Cys residues in duck Cu,Zn SOD.     

Purification of the Cytosolic CuZn-Superoxide Dismutase (CuZn-SOD) of Marachantia paleacea var. diptera and its Resemblance to CuZn-SOD from Chloroplasts

Suspension-cultured cells of Marchantia paleacea var. dipteracontain a single form of CuZn-superoxide dismutase (SOD; EC1.15.1.1 [EC] ) which is localized in the cytosol. SOD activity wasfound in cells cultured under heterotrophic, photoheterotrophicand photoautotrophic conditions. The CuZn-SOD was purified tohomogeneity from liverwort cells that had been cultued hetertrophically.Its molecular mass was 32.6 kDa, and it contained 17.5 kDa subunits,an indication that the enzyme is a homodimer. The enzyme hadpeaks of absorption at 252, 258 and 264 nm in the ultravioledregion, due to the presence of phenylalanine, and a peak at680 nm in the visible region, which is characteristic of CuZn-SODsfrom cholorplasts. The amino acid sequence of the amino-terminalregion of the enzyme exhibited a very high degree of homologyto those of cholorplast CuZn-SODs. An antiserum raised againstthe CuZn-SOD from liverwort cross-reacted more strongly withthe enzyme from spinach chloroplasts, than with the enzyme fromspinach cytosol. These results indicate that the CuZn-SOD ofliverwort resembles CuZn-SOD in chloroplasts even though theformer is located in the cytosol. (Received November 27, 1995; Accepted April 5, 1996)     

近江牡蛎铜锌超氧化物歧化酶的纯化及部分性质研究

经６５℃加热,硫酸铵分级沉淀,ＳｅｐｈａｄｅｘＧ－１００凝胶过滤和ＤＥ－５２柱层析,从近江牡蛎（ＯｓｔｒｅａｒｉｖｕｌａｒｉｓＧｏｕｌｄ）软体部分提纯了铜锌超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）．对其理化性质鉴定表明,用此法纯化的酶纯度均一．该酶系由两个相同亚基组成的二聚体,分子量２７．９ｋＤ．该酶的紫外吸收峰在２７２．５ｎｍ,红外光谱表现出其氨基酸组成特征,与猪血ＳＯＤ存在差异．该酶在不同的升温速率下及经不同浓度的Ｈ２Ｏ２处理后的稳定性与猪血ＳＯＤ不同．其氨基酸组成与不同来源的同类酶存在差异．     

DNA cleavage mediated by copper superoxide dismutase via two pathways

The known action of Cu, Zn superoxide dismutase (Cu(2)Zn(2)SOD) that converts O(2)(-) to O(2) and H(2)O(2) plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of Cu(2)Zn(2)SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu(2)Zn(2)SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu(n)SOD that contains 1-4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form of Cu(2)Zn(2)SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu(n)SOD is as active as the Cu(II) in the copper site of fully active Cu(2)Zn(2)SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu(n)SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only <5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu(n)SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu(n)SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu(n)SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu(2)Zn(2)SOD and its mutants) might have the DNA cleavage activity.