羊红细胞铜锌超氧化物歧化酶的纯化及部分性质研究

目的从羊红细胞中分离纯化铜锌超氧化物歧化酶 (Cu ,Zn SOD) ,并对其部分理化性质进行研究。方法采用有机溶剂去除血红蛋白、热变性、超滤浓缩、丙酮沉淀、DE FF柱色谱的方法 ,对羊红细胞Cu ,Zn SOD进行分离纯化。结果羊红细胞 5 2 0g得Cu ,Zn SOD总活力为 4 6 70 0 0u ,比活为 812 6u/mg·pro,纯化倍数为 2 6 .2 ,活性回收率为 6 1%。理化性质分析表明 :该酶的最大紫外吸收波长为 2 5 8nm ,亚基相对分子量为 16 .1kD ,每个亚基含 1个铜原子和 1个锌原子。pH在 6～ 11范围内该酶稳定性很好 ,在 35℃～ 75℃范围内 ,保温 2 0min ,酶活基本没有损失。 2mmol/L的SDS对Cu ,Zn SOD没有明显的抑制作用 ,而 2mmol/L的H2 O2 对该酶表现出较强的抑制作用。结论采用此方法分离纯化的Cu ,Zn SOD达到电泳纯 ,其理化性质与其它动物血液来源的Cu ,Zn SOD一致。     

柞蚕幼虫铜锌超氧化物歧化酶的纯化及部分性质研究

目的从柞蚕幼虫分离铜锌超氧化物歧化酶 (Cu ,Zn SOD) ,并对其部分性质进行研究。方法用氯仿 乙醇除杂蛋白质、丙酮沉淀、热变性、DE 32柱色谱等方法 ,对柞蚕幼虫Cu ,Zn SOD进行分离纯化。结果得Cu ,Zn SOD干粉 9.5 3mg ;比活 10 116 .5 8u/mgpro ;活力回收 5 5 .87% ;纯化倍数为 36 0 .6 7。该酶相对分子质量 (Mr)约为 310 0 0 ;亚基Mr 约为 15 5 0 0 ;最大吸收波长 2 5 8nm ;pH在 5～ 9范围内基本稳定 ;在 4 0℃～ 6 0℃内保温 30min酶活基本不变 ;2mmol/L十二烷基硫酸钠对此酶没有明显的抑制 ;该酶对KCN和H2 O2 敏感。结论柞蚕幼虫含有丰富的Cu ,Zn SOD。     

羊红细胞铜锌超氧化物歧化酶的纯化及部分性质研究

目的：从羊红细胞中分离纯化铜锌超氧化物歧化酶(Cu，Zn—SOD)．并对其部分理化性质进行研究．方法：采用有机溶剂去除血红蛋白，热变性，超滤浓缩，丙酮沉淀、DF—FF柱色谱的方法．对羊红细胞Cu．Zn-SOD进行分离纯化。结果：羊红细胞520g得Cu，Zn—SOD总活力为467000IU．比活为8126μ／mg.pro，纯化倍数为26．2，活性回收率为61％，理化性质分析表明，该酶的最大紫外吸收波长为285nm，亚基相对分子量为16．1ku，每个亚基含1个铜原子和1个锌原子，pH在6-11范围内该酶稳定性很好，在35℃-75℃范围内，保温20min，酶活基本没有损失。2mmol／L的SDS对Cu，Zn—SOD没有明显的抑制作用，而2mmol／L的H2O2对该酶表现出较强的抑制作用，结论：采用此方法分离纯化的Cu，Zn—SOD达到电泳纯，其理化性质与其它动物血液来源的Cu，Zn—SOD一致。     

异亚丙基莽草酸对大脑中动脉栓塞大鼠脑组织自由基代谢的影响

目的　研究异亚丙基莽草酸 (ISA)对大脑中动脉栓塞模型 (MCAT)大鼠脑组织自由基代谢的影响。方法　采用三氯化铁致大脑中动脉栓塞模型 ,观察ISA对脑组织总超氧化物歧化酶 (T SOD)、Cu Zn SOD、谷胱甘肽过氧化物酶(GSH px)活力及丙二醛 (MDA)含量的影响。 结果　ISA2 0 0 ,10 0mg·kg-1可提高模型大鼠脑组织T SOD活力 ;ISA2 0 0 ,10 0 ,5 0mg·kg-1均可明显增加模型大鼠脑组织Cu Zn SOD和GSH px活力 ;ISA 2 0 0 ,10 0 ,5 0mg·kg-1对模型大鼠脑组织MDA升高有明显的抑制作用。结论　ISA可能通过减轻脑组织自由基损伤保护脑组织缺血性损伤     

豆乳凝固酶产生菌LD30的研究

目的从土壤中分离产豆乳凝固酶菌株,并研究其发酵条件和酶学性质。方法采用常规土壤分离方法和培养基。结果从广东、敦煌、吐鲁番等地得到的12份土壤样品中分离得到1株产豆乳凝固酶的菌株芽孢杆菌LD30。在给定的发酵条件下,豆乳凝固酶的产率达到600 U.L-1。酶学性质表明:该酶的最适反应温度为70℃,60℃保温1 h,残余酶活力为50%。在pH6.0~7.0内,酶活力稳定,最适酶反应的pH值为5.9。结论筛选到的产豆乳凝固酶菌株芽孢杆菌LD30具有较高的产酶活性,值得进一步深入研究。     

葡萄糖和Cu2+对蛹虫草Cu,Zn超氧化物歧化酶在E.coli中表达的影响

对含有蛹虫草Cu，Zn-超氧化物歧化酶（cm—SOD）基因的重组E．coli培养条件进行了初步优化。结果表明，LB培养基中含有Cu^2＋可明显提高超氧化物歧化酶比活力；而在菌体培养中补加葡萄糖，可提高重组蛋白表达量和细胞生物量。在含有0．6mmol／L Cu^2＋、Zn^2＋的LB培养基中补加22mmol／L葡萄糖，每200ml培养液可获得1g湿细胞，超氧化物歧化酶比活力达到3305u／mg。     

假单胞菌发酵制备精氨酸脱亚胺酶

采用假单胞菌（Pseudomonas sp．）发酵制备精氨酸脱亚胺酶，优化发酵培养基配方及培养条件，以葡萄糖为碳源，酵母膏和蛋白胨为氮源，30℃振荡培养20h时，发酵液中精氨酸脱亚胺酶的酶活力可达2．17u／ml。向HepG2肝癌细胞培养液中加入酶粗品0．5u／ml，对肿瘤细胞的生长抑制率为93．4％。     

黏质沙雷氏菌产几丁质酶二步发酵工艺的优化

采用二步发酵法对…株黏质沙雷氏菌（Serratia marcescens）的产几丁质酶发酵条件进行研究,以期在已优化的一步发酵的基础上酶活力得到进一步提高。在二步发酵产酶的过程中,通过单因素法优化得到二步发酵培养基的最适碳源、氮源浓度和菌龄,同时选取发酵时间、初始pH、接种菌体量和装液量4个因子进行正交试验。最终得到的最优条件为：胶体几丁质0．8％,酵母粉1．0％,菌龄12h,发酵时间72h,初始pH8．5,菌体干重90mg,装液量20mL。在此条件下,酶活力可达1．021U·mL^-1。在二步发酵工艺中另添加0．1％纤维素,酶活力可提高至1．128U／mL,比一步发酵提高了1．27倍。     

酵母菌乳糖酶中试制备工艺研究

研究了酵母菌乳糖酶中试制备工艺包括发酵、菌体破壁、酶液精制等。以2％乳糖、2％葡萄糖、2％玉米浆、0．5％酵母膏、1．5％尿素配方作为发酵培养基，通过对发酵各参数的优化控制，在500L规模发酵罐中进行发酵，单位体积酶活达13．8U／ml。此外，还建立了较大规模酵母菌细胞壁快速破壁方法，以及简便的乳糖酶提取制备工艺。整套工艺操作简便，成本低廉且无须添加任何大型设备，酶的总提取收率在67％以上。该酶制品在6℃低温条件下可在较短时间内有效水解乳制品中的乳糖,降解率达70％以上。     

重组胸苷磷酸化酶在大肠杆菌中的表达和1-（2-脱氧-β-D-呋喃核糖基）-1，2，4-三唑-3-甲酰胺的生成

研究了大肠杆菌BL21（DE3）中重组胸苷磷酸化酶的最佳诱导表达条件及1-（2-脱氧-β-D-呋喃核糖基）-1,2，4-三唑-3-甲酰胺（1）的最佳生成条件。菌体培养3．5h时加入8mmol／L乳糖诱导6h后，胸苷磷酸化酶的酶活力为2．14×10^4u／mg湿菌体。5％湿菌体在pH7．0的0．1mol／L Tris—HCl缓冲液中与底物胸苷、1，2，4-三唑-3-甲酰胺于50℃反应5h后，所得1的转化率大于90％。     

Selective inhibition of the mouse brain Mn-SOD by methylmercury

Changes in mRNA levels, protein contents and enzyme activities for brain Cu,Zn- and Mn-SOD by methylmercury chloride (MMC) administration, were examined, over a period of 12 days in ICR male mice. After subcutaneous administration of MMC (10 mg/kg) to mice, brain mercury content reached a maximum at 2 days and remained at that level for at least 5 days. MMC exposure resulted in a time-dependent decrease in the Mn-SOD activity: the enzyme activity at 5 days after exposure to MMC was about 60% of control level whereas this exposure was without effect on the Cu,Zn-SOD activity, indicating differential sensitivity of SOD isozymes to the metal. However, levels of mRNA and protein synthesis for Mn-SOD were unaffected by MMC administration. The direct effect of MMC on the both SOD activities were further examined with purified enzyme preparations. After each SOD isozyme (10 U) was incubated with 0.2 mM MMC for 24 h at pH 7.8, the enzyme activities for Cu,Zn- and Mn-SOD were 90% and 37% of control, respectively. Incubations at a ratio of SOD to MMC (1 : 600) for 24 h resulted in a substantial decrease in the enzyme activity of the Mn form; this isozyme-selective inactivation was noted at alkaline pH. A combination of isoelectric focusing-agarose gel electrophoresis (IEF-AGE) and synchrotron radiation X-ray fluorescence (SR-XRF) analysis revealed that Mn-SOD rather than Cu,Zn-SOD underwent modification. Furthermore, a decrease in native form of Mn-SOD protein after MMC exposure was confirmed by gel filtration chromatography. These results indicate that Mn-SOD, but not Cu,Zn-SOD, is susceptible to modification by MMC and the resulting alteration in structure appears to cause a loss of enzyme activities.     

Characterization of an inducible isoform of the Cu/Zn superoxide dismutase in the blue mussel Mytilus edulis

Aerobic organisms are protected against oxidative stress by antioxidant systems which mobilise enzymes such as the Cu/Zn superoxide dismutase (Cu/Zn-SOD) which transfers O2(.-) to H2O2. In this paper, we report the characterization of three isoforms of Cu/Zn-SOD in the blue mussel Mytilus edulis and we show that one of these isoforms is strongly inducible. Cytosolic extracts of digestive gland and gills from adult blue mussels were analysed by polyacrylamide gel electrophoresis or isoelectric focusing followed by in situ staining for SOD activity. Two main bands of Cu/Zn-SOD were obtained at pI 4.7 and 4.6 corresponding to native apparent molecular weight values of 205 and 155 kDa. Blue mussels from chemically contaminated area in Le Havre harbour exhibited a third Cu/Zn-SOD isoform characterized by a more acidic isoelectric point (pI 4.55) and a native apparent molecular weight of 130 kDa. When maintained in clean marine water, mussels from this area showed a transitory decrease in total SOD activity accompanied by the disappearance of the SOD-3 band. Conversely, the exposure (4 and 8 h, and 3 and 7 days) of control blue mussels to copper (25 microg l(-1)) markedly increased SOD-3 band while the total SOD activity did not systematically change. Taken together our results suggest that the variations of SOD expression pattern in Mytihus edulis could be used as a tool for the marine environment monitoring.     

大豆超氧化物歧化酶的分离和鉴定

采用磷酸盐缓冲液使样品匀浆,两次硫酸铵分级沉淀和DEAE-Cellulose离子交换层析从大豆中分离出超氧化物歧化酶。分离酶的聚丙烯酰胺凝胶电泳呈一条蛋白谱带。过氧化氢和乙醇-氯仿试验表明,该酶属铜锌超氧化物歧化酶。酶的比活力为4710u/mg Pr,活力回收率23％,紫外吸收峰在265.4nm,分子量为32.4kD。     

Comparative study of superoxide dismutase in normal and hereditary cataract (UPL) rats

In the present study, the levels of SOD activity and Cu, Zn-SOD mRNA in the brain, kidney, liver and eye of normal and Upjohn Pharmaceutics Limited (UPL) rats, a new hereditary cataract model derived from Sprague-Dawley rats, were measured. Although the levels of SOD activity in the eye and brain of UPL rats were significantly decreased compared with those of normal rats 3 and 5 weeks after birth, the levels of SOD activities in the kidney and liver were the same in both groups. The levels of Cu, Zn-SOD mRNA in kidney and liver of UPL rats were the same as those of normal controls. The level of Cu, Zn-SOD mRNA in the brain of normal rats 5 weeks after birth was about twofold greater than that of UPL, and that in the eye of UPL rats 3 weeks after birth was significantly decreased compared with that of normal controls. The sequences of cDNA encoding Cu, Zn-SOD and the sequences of the regulatory region of the Cu, Zn-SOD gene were confirmed to be the same in normal and UPL rats. These results indicated that the decreases in levels of SOD activity and Cu, Zn-SOD mRNA in the brain and eye of UPL rat were not due to mutation of the genomic Cu, Zn-SOD gene in UPL rats or differences in the sequence of the regulatory region of the Cu, Zn-SOD gene between normal and UPL rats.     

甘糖酯抗氧化作用的分子机制

目的探讨甘糖酯(PGMS)清除自由基抗氧化作用的分子机制。方法给大鼠ig高脂乳剂建立高脂血症模型,分成对照组、甘糖酯治疗组、甘糖酯+DDC组,治疗3周。检测血清、肝脏、脾脏和主动脉中丙二醛(MDA)的含量,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)的活性,以及Cu,Zn-SOD mRNA的表达水平。结果甘糖酯治疗组大鼠,丙二醛(MDA)含量降低,SOD,GSH-Px和CAT的活性显著升高,Cu,Zn-SOD mRNA的表达水平增加;而甘糖酯和DDC联合用药治疗组,DDC抑制了甘糖酯诱导的Cu,Zn-SOD mRNA表达水平和SOD活性的升高,造成MDA含量的相应升高。结论甘糖酯通过诱导抗氧化酶SOD,GSH-Px和CAT的活性,增加Cu,Zn-SOD mRNA的表达水平,清除体内过多的氧自由基,达到抗氧化的目的。     

Downregulation of superoxide dismutase activity and gene expression in cultured rat brain astrocytes after incubation with vitamin C

Reactive oxygen species have been linked with neuropathological changes in the central nervous system. Epidemiological studies supported the beneficial effect of supplementation of antioxidants. Superoxide dismutase (SOD) is an endogenous enzyme which can scavenge reactive oxygen species. This study investigated the effect of supplementation with ascorbic acid (vitamin C) on the changes of SOD in cultured neurological cells. Rat brain astrocytes (RBA-1 cells) were incubated with vitamin C and divided into four groups: a control group (without vitamin C) and three treatment groups with vitamin C at 40, 80, and 160 micromol/l. After short-term (2 days) and long-term (7 days) incubation, SOD activity, SOD mRNA level by Northern blotting, and SOD protein amounts by Western blotting were measured. After 2 days of incubation, vitamin C resulted in a decrease in the activity of SOD in a concentration-dependent manner (Mn-SOD from 14.8 +/- 1.2 to 13.2 +/- 0.5 U/mg protein and Cu/Zn-SOD from 64.8 +/- 1.2 to 51.7 +/- 0.9 U/mg protein; p < 0.05), and vitamin C also attenuated the Cu/Zn-SOD mRNA level from 100 to 86.3 +/- 6.7%; p < 0.01), whereas the protein amounts of these two SODs remained unchanged. After 7 days of incubation with vitamin C, the SOD activity of RBA-1 cells decreased significantly (Mn-SOD from 14.9 +/- 0.3 to 11.8 +/- 0.3 U/mg protein and Cu/Zn SOD from 61.8 +/- 1.8 to 54.6 +/- 0.9 U/mg protein; p < 0.01), and the mRNA level was also attenuated (Mn-SOD from 100 to 86.8 +/- 8.7% and Cu/Zn-SOD from 100 to 84.7 +/- 4.8%; p < 0.01). These results suggest that 2 and 7 days of incubation with relatively high concentrations of vitamin C may downregulate activity and gene expression of SOD in cultured RBA-1 cells.     

肾上腺皮质疾病患者超氧化物歧化酶和丙二醛的表达及意义

目的 探讨超氧化物歧化酶活性（SOD）和丙二醛水平（MDA）在肾上腺皮质疾病患者外周血血清和组织中的表达及与意义. 方法 选择肾上腺皮质增生患者12例、肾上腺皮质腺瘤患者10例,健康对照者11例,分别比较对照组和病例组患者术前血清学铜/锌超氧化物歧化酶（Cu/Zn-SOD）、锰超氧化物歧化酶（Mn-SOD）活性和MDA含量、肾上腺组织免疫组化染色Cu/Zn-SOD、Mn-SOD阳性细胞占细胞总数百分比之间的差异. 结果 病例组各组患者术前血清中Cu/Zn-SOD、Mn-SOD活性显著低于健康对照组（P＜0.05）,MDA含量显著高于健康对照组（P＜0.05）;Cu/Zn-SOD染色面积在肾上腺皮质腺瘤组中表达显著降低（P＜0.05）,Mn-SOD染色面积在肾上腺皮质增生和肾上腺皮质腺瘤组中表达显著降低;SOD的血清学与组织学之间表达呈正相关（P＜0.05）. 结论 SOD和MDA在肾上腺皮质疾病患者血清和组织中表达异常,联合检测SOD、MDA可能为肾上腺皮质疾病的鉴别诊断提供新的方法.