蜂王浆超氧化物歧化酶分离纯化及部分性质研究

本文采用硫酸铵盐析、SephedexG-150柱层析从蜂王浆中分离出了超氧化物歧化酶（SOD）。经聚丙烯酰胺凝胶电泳显示单一蛋白区带。此酶比活力为1887 .4U/mg,纯化倍数为524.3,回收率为12.47%,最适pH8.3,最适温度为25℃。Mn^2,Cu^2 ,Ba^2 ,Mg^2 对此酶在有激活作用,Zn^2 则具有抑制作用。过氧化氢和乙醇-氯仿实验表明：蜂王浆中的SOD属Cu/Zn-SOD。     

马铃薯酪氨酸酶的性质

用硫酸铵分级沉淀、DEAE SepharoseFF和SephadexG 150柱层析,从马铃薯中分离纯化出酪氨酸酶,纯化倍数50,比活170.03U mg蛋白。SDS PAGE检测呈现一条带。分子量42KD。酶的最适pH7 5,最适温度30℃。该酶具有单酚酶和二酚酶活性,其中以邻苯二酚为底物活性最高,以L DOPA为底物Km值为3 70mmol·L-1。Hg2+、Zn2+、Fe2+为激活剂,K+、Ga2+、Mg2+是抑制剂。Ga2+为非竞争性抑制,抑制常数ki为116 8mmol·L-1,K+为竞争性抑制,抑制常数ki为10mmol·L-1。     

淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质

分离得到产抗菌聚氨基酸ε-聚赖氨酸菌株淀粉酶产色链霉菌TUST2, 从中纯化了ε-聚赖氨酸降解酶, 并对其性质进行了研究. 结果表明, 该酶为膜结合蛋白. 为提取该降解酶, 先收集菌体细胞并用超声波破碎, 细胞膜部分用1.0 mol/L NaSCN溶液溶解. 将粗酶液进行Sephadex G100凝胶柱层析分离. 用100 mmol/L磷酸缓冲液洗脱, 收集活性部分. 纯化后的样品用SDS-PAGE检测, 酶亚基分子量约为54700. 酶活力在pH=6.0~9.0间稳定, 最适宜pH=7.0. 酶的最适温度为30 ℃, 在10～50 ℃水浴30 min酶活力未见明显下降. 研究了不同金属离子对酶活力的影响, 结果表明, Zn²⁺, Cu²⁺和Fe³⁺可分别提高酶活力29.72%, 15.85%和15.08%; 但Ag⁺, Hg²⁺, Co²⁺和Mn²⁺对酶活力有强烈的抑制作用. Ca²⁺, K⁺和Ba²⁺对酶活力没有影响. 添加4%Tween-80能提高酶活力10%, 但EDTA能强烈抑制酶活力. 研究结果表明, 此降解酶的性质与白色链霉菌产生的ε-聚赖氨酸降解酶的性质相似.     

金边龙舌兰超氧化物岐化酶的提取与特性研究

金边龙舌兰经匀浆、丙酮沉淀、葡聚糖凝胶过滤等步骤,纯化获得2个超氧化物歧化酶(SOD)I和Ⅱ。该SODI和Ⅱ适应pH范围分别在4~11和4~10,pH分别为7和8时活性最高;均在60℃以下稳定,60℃能够保持较高的活力;分别在272.5和274.5 nm处有最大紫外光吸收;敏感性抑制试验证明,其活性均被氰化钾和过氧化氢所抑制,但不受氯仿-乙醇影响,与Cu·Zn-SOD相似。     

羊肝源穿山龙薯蓣皂苷-α-L-鼠李糖苷酶的分离及其动力学特性

对羊肝中含有的水解穿山龙薯蓣皂苷鼠李糖糖基的穿山龙薯蓣皂苷-α-L-鼠李糖苷酶进行了分离、纯化, 并对其动力学特性进行了研究. 结果表明, 粗酶液经DEAE-Cellulose离子交换层析柱纯化后, 其比活提高了19.9倍. 在pH＝6.8、反应温度为42 ℃、反应时间为8 h和底物浓度为23 mmol/L的条件下, 该酶达到其最高活力. 在10—200 mmol/L范围内, Fe3+和Cu2+对酶活力有明显的抑制作用, Mg2+和Zn2+对酶活力有微弱的激活作用, 而Ca2+对酶有较强的激活作用. 采用SDS-PAGE方法测得酶蛋白分子量为71000. 选择穿山龙薯蓣皂苷、人参皂苷Re和芦丁为酶反应底物, 进行酶的底物专一性研究发现, 穿山龙薯蓣皂苷-α-L-鼠李糖苷酶对其底物具有高度专一性.     

淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质

分离得到产抗菌聚氨基酸--ε-聚赖氨酸菌株淀粉酶产色链霉菌TUST2,从中纯化了ε-聚赖氨酸降解酶,并对其性质进行了研究.结果表明,该酶为膜结合蛋白.为提取该降解酶,先收集菌体细胞并用超声波破碎,细胞膜部分用1.0 moL/L NaSCN溶液溶解.将粗酶液进行Sephadex G100凝胶柱层析分离.用100mmol/L磷酸缓冲液洗脱,收集活性部分.纯化后的样品用SDS-PAGE检测,酶亚基分子量约为54700.酶活力在pH=6.0～9.0间稳定,最适宜pH=7.0.酶的最适温度为30℃,在10～50℃水浴30 min酶活力未见明显下降.研究了不同金属离子对酶活力的影响,结果表明,Zn~(2+),Cu~(2+)和Fe_(3+)可分别提高酶活力29.72%,15.85%和15.08%;但Ag~(+),Hg~(2+),Co~(2+)和Mn~(2+)对酶活力有强烈的抑制作用.Ca2~(2+),K~+和Ba~(2+)对酶活力没有影响.添加4%Tween-80能提高酶活力10%,但EDTA能强烈抑制酶活力.研究结果表明,此降解酶的性质与白色链霉菌产生的ε-聚赖氨酸降解酶的性质相似.     

自养性大豆根瘤菌膜结合态氢酶的提纯及某些特性的研究

本文证明在提纯白养性大豆根瘤菌膜结合态氢酶过程中,为了保持酶活性和防止酶蛋白的沉淀,必须排除O_2、加入还原剂和去垢剂Genapol。纯化的氢酶在H_2气氛下储存于—80℃,半年后仍保留78％的活性,最纯的氢酶其比活性为142μmol亚甲蓝还原·min~(-1)·mg蛋白~(-1)。纯化的氢酶经SDS凝胶电泳呈现二条主要蛋白带,其分子量分别接近于60000和30000。不同提纯阶段的氢酶其比活性与细胞色素b的含量不成正相关。最高比活性的氢酶不含细胞色素b。文中还讨论了自养性大豆根瘤菌膜结合态氢酶的其它特性。     

绿豆胰蛋白酶抑制剂的研究——Lys活性碎片的还原及重氧化

本文证明绿豆胰蛋白酶抑制剂Lys活性碎片由 A_1,A_2两条肽链通过两对二硫键联结而成,今通过DTT还原及凝胶过滤可将A_1(26个氨基酸残基),A_2(9个氨基酸残基)两肽链拆分.还原型的A_1肽链重氧化后活力的回收率是原有活性碎片总活力的25%.经固相胰蛋白酶亲和层析纯化的A_1活性碎片以单体和二聚体两种分子形式存在,两者可在Sephadex G-25柱上分开,以单体为主,二聚体活性较低.此结果进一步证实,绿豆胰蛋白酶抑制剂的分子进化过程是由一原始单头抑制剂通过基因重复及基因突变演化而来。此外比较了Lys活性碎片和重氧化肽链A_1的CD图谱。     

SOD1抑制与活性氧信号转导的调控

铜锌超氧化物歧化酶（Copper-zinc Superoxide Dismutase,SOD1）是胞内广泛存在的抗氧化酶,催化O₂^·-歧化为H₂O₂和O₂,维持胞内活性氧物种（ROS）内稳态。癌细胞的生长、增殖都依赖于适度高浓度的H₂O₂,高表达的SOD1维持癌细胞内较高水平的H₂O₂和ROS内稳态。抑制癌细胞内SOD1的活性,可以有效调节ROS信号通路,抑制癌细胞的生长、增殖,阻滞癌细胞周期,促进癌细胞凋亡。因此,通过抑制其活性调控癌细胞内ROS信号网络并选择性杀死癌细胞已经成为抗癌药物设计的一个重要方向。本文主要总结了各类SOD1抑制剂,以及SOD1抑制对ROS信号转导的调控,分析了SOD1特异抑制选择性杀死癌细胞的机制。     

碘离子胁迫对绿豆萌发过程4种微量元素的影响

用火焰原子吸收法测定绿豆萌发过程中铜、铁、锰和锌的含量,研究不同含量碘离子胁迫对绿豆萌发过程中生理活动的影响。结果显示,各含量培养液所培养的绿豆芽苗中4种元素含量随培养时间的增加而显著降低,适当质量浓度的碘离子(20~80 mg/L)可以促进绿豆对铜、铁、锌的溶出,但碘离子质量浓度过高(100 mg/L)时将抑制绿豆对铜、铁、锰的溶渗出,培养末期绿豆均能从培养液中重新利用铁、锰、锌3种微量元素。     

Zinc deficient bovine erythrocyte superoxide dismutase has low specific activity.

Zinc deficient bovine superoxide dismutase (Cu2E2SOD (E = empty)) was prepared and purified by high performance liquid chromatography (HPLC). Each peak was characterized as to protein, copper content and specific activity. The Cu2E2SOD peak fractionated by HPLC has a low specific activity at pH 7.8 (about 10% of the native enzyme (Cu2Zn2SOD)). With the addition of zinc ions, the specific activity of Cu2E2SOD was quantitatively restored to that of the native enzyme. This behavior implies that the zinc ion is very important for the appearance of enzyme activity.     

Identification of different classes of superoxide dismutase in carnation petals

Isoelectric focusing and nondenaturing two-dimensional electrophoresis, followed by an enzymatic characterization involving use of specific inhibitors were applied to identify the different forms of the superoxide dismutase (SOD) extracted from petals of cut carnation. Electrophoresis was carried out either with the crude extract or with fractions obtained after ion exchange chromatography of the extract. Three forms of the SOD were identified (Cu,Zn-SOD, Fe-SOD and Mn-SOD). They have close pI's (4.7 to 4.9) and most of them are composed of several isoforms.     

Purification and Characterization of a Cu,Zn-SOD from Garlic (Allium sativum L.). Antioxidant Effect on Tumoral Cell Lines

Crude garlic extract contains one Mn-superoxide dismutase designated as SOD1 and two Cu,Zn superoxide dismutases as SOD2 and SOD3. The major isoform SOD2 was purified to homogeneity by Sephacryl S200-HR gel filtration, DEAE Sepharose ion exchange chromatography, and chromatofocusing using PBE 94. SOD2 was purified 82-fold with a specific activity of 4,960 U/mg protein. This enzyme was stable in a broad pH range from 5.0 to 10.0 and at various temperatures from 25 to 60°C. The native molecular mass of SOD2 estimated by high performance liquid chromatography on TSK gel G2000SW column was 39 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 18 kDa, suggesting that native enzyme was homodimeric. The isoelectric point as determined by chromatofocusing was 5. Analysis of its N terminal amino acid sequence revealed high sequence homology with several other cytosolic Cu,Zn-SODs from plants. Exposure of cancer cell lines to garlic Cu,Zn-SOD2 led to a significant decrease in superoxide content with a concomitant rise in intracellular peroxides, indicating that the enzyme is active in mammalian cells and could, therefore, be used in pharmacological applications.     

Purification of the membrane protein enzyme lipoamidase by affinity chromatography

Lipoamidase, a membrane glycoprotein enzyme, was purified from brain membrane by means of various affinity columns. A column with immobilized Arg-Phe-NH2 was found to be the most effective. After loading the crude material of the membrane, and extensive washing of the column with sodium chloride (0.3 M) solution, the enzyme activity was eluted by a solution containing 1% of nonionic detergent (Nonidet P-40). The fractions containing the lipoamidase activity were analyzed by SDS-PAGE, and a single protein band detected in this fraction. On the other hand, lipoyl-affinity columns with various resins were not effective in enzyme purification. Single step chromatography on the Arg-Phe-NH2 column enriched the membrane enzyme lipoamidase by 40-fold. The mechanism by which this affinity resin effectively enriches the enzyme remains to be elucidated.     

Comparative superoxide dismutase activity studies

Our results indicate that the various methods for measurement of superoxide dismutase (SOD) activity differ considerably as regards sensitivity. The activity difference may be particularly striking in the determination of the SOD activities of crude homogenates, because of the large number of disturbing factors. We consider the epinephrine-adrenochrome method to be the most suitable for measurement of the SOD activities of crude homogenates and purified enzyme preparations.     

固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶

以Ｃｕ~（２＋）－Ｓｅｐｈａｒｏｓｅ４Ｂ固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶,考察了不同的洗脱缓冲溶液及其ｐＨ对纯化效率的影响,显示了方法的有效性。     

A magnetizable solid phase for enzyme extraction

A method for the convenient and reliable preparation of magnetizable agarose beads containing iron particles is described. The beads were treated with the triazine dye, Reactive Red 120, and the matrix was examined for the ability to extract proteins from crude preparations using lactate dehydrogenase from porcine muscle as a model. The recovery and specific activity values of enzyme obtained using this matrix and magnetic field separation were significantly greater than those for enzyme purified by centrifugation and conventional dye ligand chromatography.     

Purification and Characterization of Superoxide Dismutase （SOD） from Camellia Pollen

A superoxide dismutase（ SOD ） was purified to homogeneity from fresh camellia pollen by means of ammonium sulfate precipitation and column chromatography with DEAE-cellulose（ DE52 ）, Sephadex G-100 and phenyl sepharose^TM 6 Fast Flow columns. Its specific activity could reach to 4034 U/mg protein and it was determined to be Cu/ Zn-SOD according to its different sensitivities to different inhibitors. The molecular weight of the SOD and its subunit were 69500 and 34700, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）, which implicates that the SOD in camellia pollen is a dimmer composed of two identical subunits. The isoelectric point of the enzyme was determined to be 4. 1 by isoelectric focusing electrophoresis and the N-terminal amino acid was identified to be Gly by the DNS-Cl method. Its α-Helix was also calculated to be approximately 21.8% according to the circular dichroism（CD） spectra.     

PS胶体粒子表面逐层自组装固定化SOD及其生物活性

通过逐层自组装技术成功地把超氧化物歧化酶(SOD)吸附在聚苯乙烯(PS)胶体粒子表面.zeta电位和TEM证明了聚阳离子或聚阴离子型SOD与相反电荷的聚电解质在PS胶体粒子表面的交替吸附.通过测定SOD被胶体粒子吸附后上清液的生物活性,得到聚阴离子型SOD(pH=8.0)和聚阳离子型SOD(pH=4.3)在PS胶体粒子表面的吸附量分别为12和51IU,相对活性分别为23.4%和2.9%.聚阴离子型SOD在PS胶体粒子表面能形成平滑规整的膜,导致较高的相对活性.研究结果表明,通过调节pH值,可以优化自组装固定化酶的聚集状态和生物活性     

无花果叶超氧化物歧化酶的分离、纯化及性质研究

以我国山东无花果树(Brunswike, 原产法国Ficus carica)树叶为原料, 采用缓冲液抽提、硫酸铵分级、DEAE-Sepharose Fast Flow阴离子交换层析和Sephacryl S-100 HR分子筛层析分离纯化得到电泳纯CuZn-SOD, 并对其酶学性质进行研究.