内切酶SCaI对不同纯度质粒的酶解效率探讨

限制性内切酶ＳＣａＩ适用于多种质粒载体,尤其是融合蛋白表达载体,ｐＧＥＸ系统,因它具有二个ＳＣａＩ酶切位点,因此用ＳＣａＩ酶解图谱鉴定以ｐＧＥＸ系统为载体的重组子甚为便捷。简便碱裂解法提取重组ＤＮＡ省时,省耗。本文探讨了ＳＣａＩ对用常规和简便两种碱裂解法提取的重组ＤＮＡ的酶解效率。结果表明：（１）简便碱裂解法抽提的重组ＤＮＡ,用ＥｃｏＲＩ、ＥｃｏＲＶ、ＰｓｔＩ、ＢａｍＨＩ等限制性内切酶酶解,可以获     

青霉胞外菊粉酶的纯化和性质研究

青霉菌（ＰｅｎｉｃｉｌｌｉｕｍＳＰ．９１－４）产生的胞外菊粉酶（ｅｘｔｒａｃｅｌｌｕｌａｒｉｎｕｌｉｎａｓｅ）粗酶液,经硫酸铵沉淀,超滤浓缩,Ｓｅｐｈａｄｅｘ－Ｇ－１００凝胶过滤,ＤＥＡＥ－Ｓｅｐｈａｃｅｌ离子交换柱层析等步骤,得到提纯３０倍的酶Ｅ。酶Ｅ反应时最适ｐＨ４．５,最适温度为５０℃,在ｐＨ４．７～７．６范围内,温度５０℃以下酶Ｅ活性稳定;其活性受Ａｇ＾＋,Ｃｕ＾２＋ＰＣＭＢ强烈抑制。采用     

夏威环毛蚓纤溶酶的分离纯化及部分性质研究

以夏威环毛蚓（Ｐｈｅｒｅｔｉｍａ ｈａｗａｙａｎａ）为材料,采用磷酸盐缓冲液抽提、（ＮＨ４）２ＳＯ４分段盐析,离子交换树脂 Ｄ２９０、 Ｓｅｐｈａｄｅｘ Ｇ－１００和 ＤＥＡＥ－ｓｅｐｈａｄｅｘ Ａ－５０三种连续柱层析方法得到一种在 ＰＡＧＥ上显示单一区带的纤溶酶组份。采用凝胶柱层析和 ＳＤＳ－ＰＡＧＥ测其分子量为 １２ ０００和 １２３００,由一条肽链组成。该酶具有强烈的纤溶活力和水解ＢＡＥＥ的活力,能直接作用纤维蛋白和间接激活纤溶酶原。其最适反应温度为４５℃,最适反应ｐＨ为８．０。该酶水解ＢＡＥＥ的活力可被Ｎａ＋、Ｋ＋、Ｍｇ２＋、Ｈｇ２＋、金属离子和ＥＤＴＡ、巯基乙醇抑制,Ｃａ＋则有激活作用。该酶中性糖含量为５％,氨基酸组成中Ａｒｇ、Ｌｅｎ含量较多．     

兼具SOD和GPX活力的双功能酶的制备及性质研究

用苯甲基磺酰氟（ＰＭＳＦ）和Ｈ２Ｓｅ相继处理铜锌超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）,将酶分子中的丝氨酸（Ｓｅｒ）转化为硒代半胱氨酸（ＳｅＣｙｓ）,从而引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基因,使其在ＳＯＤ酶活性大部分保留的情况下,具有ＧＰＸ活性,其ＧＰＸ活力是ＰＺ５１活力的３０倍,研究了双功能酶的最佳制备条件,包括ＰＭＳＦ的剂量、反应最适温度及Ｈ２Ｓｅ处理时间等,并用电子能谱、ＤＴＮＢ等方法     

兼具SOD和GPX活力的双功能酶的制备及性质研究

用苯甲基磺酰氟（ＰＭＳＦ）和Ｈ_２Ｓｅ相继处理铜锌超氧化物岐化酶（Ｃｕ,Ｚｎ－ＳＯＤ）,将酶分子中的丝氨酸（Ｓｅｒ）转化为硒代半胱氨酸（ＳｅＣｙｓ）,从而引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团,使其在ＳＯＤ酶活性大部分保留的情况下,具有ＧＰＸ活性,其ＧＰＸ活力是ＰＺ５１活力的３０倍。研究了双功能酶的最佳制备条件,包括ＰＭＳＦ的剂量、反应最适温度及Ｈ_２Ｓｅ处理时间等,并用电子能谱、ＤＴＮＢ等方法测定了双功能酶的硒含量;测定了双功能酶对不同底物的米氏常数及双功能酶的荧光光谱、紫外吸收光谱及稳定性。     

SELEX法筛选特异亲和淀粉的小分子RNA

ＳＥＬＥＸ法筛选特异亲和淀粉的小分子ＲＮＡ＊王琛金由辛＊＊王德宝（中国科学院上海生物化学研究所分子生物学国家重点实验室,上海２０００３１）关键词ＳＥＬＥＸ;２．５ＳＲＮＡ;兔肌糖元分枝酶指数式富集法配体进化,即ＳＥＬＥＸ［１］（ｓｙｓｔｅｍ－ａｔｉｃ...     

植物遗传多样性研究中等位基因酶分析的遗传参数及其计算方法（综述）

植物遗传多样性研究中等位基因酶分析的遗传参数及其计算方法（综述）葛学军（中国科学院华南植物研究所,广州５１０６５０）ＴＨＥＧＥＮＥＴＩＣＰＡＲＡＭＥＴＥＲＳＡＮＤＴＨＥＩＲＣＡＬＣＵＬＡＴＩＮＧＭＥＴＨＯＤＳＯＦＡＬＬＯＺＹＭＥＡＮＡＬＹＳＩＳＩＮＰ...     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

三草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＢＭ９６０２产生的中性内切β－甘露聚糖酶（ｅｎｄｏ－β－１,４－Ｄ－ｍａｎｎａｎ ｍａｎｎａｎｏｈｙｄｒｏｌａｅｓ,ＥＣ,３．２．１．７８）经硫酸铵分级沉淀、ＤＥＡＥ－纤维素（ＤＥ２２）离子交换柱层析,得到电泳纯的样品,提纯了４５．５倍,收率为５．９％。用ＳＤＳ－ＰＡＧＥ测得该酶的分子量为３５ｋＤ。用ＰＡＧＥＩＥＦ测得其等电点ｐⅠ为４．５。酶反应的     

黑曲霉ρ-葡萄糖甘酶的提纯与性质

从黑曲霉Ａｓｐｅｒｇｉｌｕｓｎｉｇｅｒ发酵液中分离提纯了β－葡萄糖苷酶。提纯步骤通过（ＮＨ４）２ＳＯ４分级沉淀,ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＳｅｐｈａｄｅｘＧ－１００等三步纯化,得到凝胶电泳均一的β－葡萄糖苷酶。该酶的最适ｐＨ４．５,最适温度６０℃,Ｋｍ为０．４４（ＮＰＧ）,并有较好的热稳定性。用ＳＤＳ－凝胶电泳法和凝胶色谱法测得该酶的分子量为１２００００     

糖尿病大鼠胰岛的一氧化氮合酶(NOS)与胰岛毛细血管三维结构的改变

以还原型辅酶Ⅱ黄递酶（ＮＡＤＰＨｄ）组织化学方法及扫描电镜（ＳＥＭ）,对正常组和糖尿病组大鼠胰岛的一氧化氮合酶（ＮＯＳ）与胰岛毛细血管的三维结构进行了观察。结果表现：正常组胰岛呈弥漫的蓝黑色ＮＯＳ阳性反应产物,但在糖尿病组胰岛的ＮＯＳ反应则明显减弱。ＳＥＭ显示正常组胰岛毛细血管蟠曲呈球状,且血管管径基本一致,而在糖尿病组胰岛中则可见部分毛细血管明显扩张。因此我们认为,糖尿病大鼠的胰岛毛细血管结构和功能均发生了改变。     

非稳态酶活化动力学的布尔函数图论分析

以非稳态酶动力学的布尔函数图形方法研究非稳态酶活化动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了酶活化反应体系的非稳态酶动力学过程．     

Ping Pong Bi Bi机制的非稳态酶动力学分布函数图论研究

以非稳态酶动力学的布尔函数图形方法,来研究一类PingPongBiBi机制的非记酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类PingPongBiBi机制酶反应体系的非稳态酶动力学方程。     

Random Bi Bi机制的非稳态酶动力学布尔函数图论研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类Random Bi Bi机制的非稳态酶动力学问题,推导同此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类Random Bi Bi机制酶反应体系的非稳态酶动力学方程。     

On true and apparent Michaelis constants in enzymology. III. Is it linear dependence between apparent michaelis constant and limiting rate and is it possible to determine the substrate constant value using this dependence?

The Slater-Bonner method which is used for graphic determination of substrate constant (Ks) by linear dependence of apparent Michaelis constant (Km(app)) on the limiting rate (V(app)) of enzyme-catalysed reactions with activator participation has been critically analysed. It has been shown that although it is possible to record the mechanisms of such reactions as a scheme similar to Michaelis-Menten model which allow to find correlation Km(app) and V(app) as equation Km(app) = Ks + V(app)/k1[E]0 ([E]0 is a total enzyme concentration, k1 is a rate constant of enzyme-substrate complex formation from free enzyme and substrate) in order to calculate Ks and individual rate constants (k1, k(-1)), but this approach for investigation of all reactions with activator participation ought not to be used. The above equation is not obeyed in general, it may be true for some mechanisms only or under certain ratios of kinetic parameters of enzyme-catalysed reactions.     

5个品系小鼠胚胎干细胞系建立的方法学比较

以70％的大鼠心脏细胞条件培养基(RH-CM)为培养液,以小鼠胚胎成纤维细胞(PMEF)为饲养层,采用添加1％鸡血清的消化液和“连续离散法”作为小鼠Es细胞建系的改进方法,比较了5个品系小鼠ES细胞系建立的特点。与常规方法相比,3个近交系小鼠129／ter、C57BL/6J、BALB／c的ES细胞建系率分别由11．8％、3．7％和2．9％提高到33．3％、13．3％和19．4％,差异十分显著;直接采用改进的方法建立KM和ICR小鼠ES细胞系,建系率分别达12％和42．1％。讨论了ICM增殖的时间,即离散时机对ES集落形成及建系率的影响,结果显示：129／ter、C57BL/6J、BALB／c、KM和ICR小鼠品系ICM适宜的离散时机分别为增殖4～6d、3～3．5d、4d、4～5d和4～5d;同时,讨论了不同ES细胞建系所需最适宜的消化液浓度,其中BALB／c小鼠的ES细胞对高浓度的消化液十分敏感,0．05％Trypsin-0．008％EDTA是其比较理想的离散浓度。设计了两种离散方法,即“一次离散法”和“连续离散法”,用来离散增殖的ICM和ICM离散后出现的ES集落,结果表明：后者在建系过程中的作用明显优于前者。RH-CM与添加uF的常规ES细胞培养基相比,不但具有显著抑制小鼠ES细胞分化、维持其二倍体核型的作用,而且明显促进ES细胞的贴壁生长。新建细胞系鉴定结果表明,这一改进方法有效地维持了其作为多能性胚胎干细胞的一系列特征。     

Isolation and culture of embryonic stem cells from porcine blastocysts

This study was conducted to establish embryonic stem (ES) cell lines from porcine blastocysts. Blastocysts were collected from China miniature pigs at day 7-9 of pregnancy. Embryos were either directly (intact embryos) cultured on mitomysin C-inactivated murine embryonic fibroblasts (MEF) as feeder layers, or were used to isolate the inner cell masses (ICM) by enzyme digestive method and then cultured. It was found that enzyme digestive method could isolate ICMs without any damages of cells in all blastocysts (28). All ICMs attached to the feeder layers. Primary cell colonies were formed in 68% of ICM culture and 28% of intact blastocyst culture. Two ES cell lines derived from ICM passed six subcultures (passages). These cells morphologically resembled mouse ES cells and consistently expressed alkaline phosphatase activity. When the ES cells were cultured in a medium without feeder layer and leukemin inhibitory factor, they differentiated into several types of cells including neuron-like, smooth muscle-like, and epithelium-like cells. Some cells formed embryoid bodies in a suspension culture. These results indicate that porcine ES cell line can be established under the present experimental conditions and these ES cells are pluripotent.     

The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis

The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.     

Application of a graphic method for analyzing the kinetics of multienzyme reactions

A new graphic method is proposed to solve kinetic equations for polyenzymic reactions. Each graph apex is corresponded by the transmitting function deduced from kinetic equations by means of Laplas transformation. Application of this procedure allows to simplify the solution of kinetic equations and its analysis. The procedure suggested makes it possible to use the methods of automatic control when solving theoretical problems of enzymology.     

一类非竞争性抑制的非稳态酶动力学布尔函数图解研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类非竞争性抑制的非稳态酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类非竞争性抑制的非稳态酶动力学的动力学过程。     

反竞争性抑制的非稳态酶动力学布尔函数图解研究

以非稳态酶动力学的布尔函数图形方法,来研究一类反竞争性抑制的非稳态酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类反竞争性制酶反应体系的非稳态酶动力学问题。