小肠平滑肌胰岛素受体的特征及其蛋白激酶活性

 本实验对狗小肠平滑肌中胰岛素受体的结构和特征进行了分析研究。通过麦胚凝集素琼脂糖和两次Sepharose-CL-6B凝胶层析从平滑肌中纯化胰岛素受体,达到电泳纯。SDS-聚丙烯酰胺凝胶电泳证明胰岛素受体是由两个亚基组成的,分子量分别为135kD和90kD。磷酸化实验证明平滑肌胰岛素受体具有胰岛素依赖性蛋白激酶活性,能催化自身的β亚基磷酸化和底物的磷酸化。Scatchard分析表明胰岛素和受体的结合呈(?)协同效应,最大结合率为13μg胰岛素/mg蛋白质。     

马铃薯AGPase大小亚基功能研究

马铃薯 1,6 二磷酸腺苷葡萄糖焦磷酸化酶 (AGPase)是淀粉合成的限速酶 ,该酶有大、小两个亚基形成异源四聚体。总结了迄今为止已克隆的马铃薯AGPase大、小亚基编码基因、小亚基和底物结合位点的识别、以及大亚基异构调控因子结合位点识别的研究结果 ,提出了大小亚基非自然重组是深入研究AGPase的途径 ,建立体内条件下高效可靠代谢调控研究手段是AGPase研究所必需的。     

蛋白磷酸酶PP2A主要通过B’家族调节亚基介导Dishevelled2蛋白的去磷酸化

Dishevelled2(Dvl2)是Wnt信号通路中的关键蛋白因子且受到剧烈的磷酸化调控。蛋白磷酸酶2A(PP2A)是Dvl2的一种磷酸酶,参与Dvl2的去磷酸化调控。PP2A有多达16种调节亚基,决定着PP2A的底物特异性,但参与调节Dvl2去磷酸化的PP2A调节亚基尚未有全面研究。该文在一种细胞系中,通过siRNA逐一敲低PP2A调节亚基基因表达,分析了所有调节亚基在Dvl2磷酸化调控中的参与程度。结果显示,多种PP2A调节亚基参与Dvl2去磷酸化,其中B’家族全部成员均有参与,起到主要调控作用。细胞共定位和蛋白互作实验结果同样印证PP2A调节亚基B’家族成员参与Dvl2蛋白的磷酸化调控。该研究明确了对Dvl2蛋白去磷酸化起调控作用的PP2A调节亚基,有助于了解PP2A调节亚基的细胞生物学功能以及与底物的关系。     

变构现象中的类似相变重整化群研究

一、引言所谓变构现象,是指寡聚蛋白(例如变构酶)这样的大分子与配位体(例如底物)这样的小分子结合中表现的协同现象,是分子生物物理中研究得很活跃的课题。如果令θ_s表示酶蛋白分子的亚基结合底物分子的百分数,θ_c表示酶蛋白分子亚基的某种构象呈现的百分数,则在热平衡条件下,实验观测到在不同配位体浓度s下,θ_s或θ_c与s的关系皆呈s-型曲线,如图1。S-型曲线表现了系统的宏观景像在某个配     

蛋白质泛素化系统

泛素化是单个或多个泛素在泛素激活酶,泛素结合酶及泛素蛋白质连接酶的作用下共价修饰底物蛋白质的过程,近年来的研究发现,许多含环指的蛋白质本身是蛋白质泛素连接酶,或是多亚基连接酶中的重要成分。由于细胞内可表达200以上的环指蛋白,并且多亚基连接酶可利用同一环指蛋白但不同的底物识别蛋白。这些研究极大地丰富了对泛素化系统酶的认识,也使进一步调节和干预连接酶与底物的相互作用成为可能,新近的研究还发现,泛素化不仅可导致蛋白质的降解,还可直接影响蛋白质的活性和细胞内定位,是调节细胞内蛋白质功能和水平的主要机制之一。     

CK2 β的蛋白表达与纯化及其RNA沉默的研究

蛋白激酶CK2是一个丝氨酸/苏氨酸特异性的激酶,通常以异源四聚体形式存在,由两个催化亚基(CK2α和CK2α′)和两个调节亚基CK2β组成。CK2激酶α亚基含有和其它蛋白激酶的催化功能域同源的激酶功能域。CK2激酶β亚基在调节a亚基的基本活性方面起着很复杂的作用。通过CK2β的结合活性,CK2α改变它的活性和作用底物。本研究成功的表达和纯化了CK2β蛋白,并且构建了CK2β的S iRNA载体,为其进一步功能研究做好准备。     

我看负协同性变构酶

我看负协同性变构酶赵登蔚（蚌埠医学院生化教研室,蚌埠２３３００３）关键词变构酶,负协同性对于天冬氨酸转氨基甲酰酶（ＡＴＣ酶）这样的酶来说,天冬氨酸既是它的底物又是变构剂,它能触发所谓同促正协同效应,即底物分子结合到酶（寡聚体）的第一个亚基所引起的构象...     

硝酸还原酶的研究——Ⅵ.水稻硝酸还原酶的纯化和特性

水稻叶片NR在Blue Dextran-Sepharose 4 B亲和层析法的纯化过程中,继100μMNADH洗脱出现一个酶活峰后,再用0.25MKNO_3洗脱又出现一个更高的酶活峰。酶与BlueDextran-Sepharose 4B有两个结合位点,它们可能就是酶与底物NADH和NO_3的结合位点。NADH能和Blue Dextran竞争与酶结合而把NR从亲和柱上洗脱下来。硝酸盐除了具一般盐类的离子强度外,还有明显的底物效应。如果将KNO_3-洗脱的NR再反复进行亲和层析,又可得到NADH-洗脱和KNO_3-洗脱的两部分NR。这两部分NR在聚丙烯酰胺凝胶电泳和SDS-聚丙烯酰胺凝胶电泳上都呈现一条相同的蛋白带。 水稻NR的全酶分子量约330Kd,亚基分子量约57Kd,故全酶可能由6个相同亚基组成,酶活的pH范围为6.5～8.5,最适pH为7.5;酶对底物NO_3-和NADH的K_m值分别为3.3×10~(-4)M和2.9×10~(-5)M。     

睫状神经营养因子受体研究进展

本综述了睫状神经营养因子受体各个亚基的分子和基因结构,对ＣＮＴＦ受体信号转寻机理进行概括,并探讨ＣＮＴＦ与造血生长因子在信号转导上的相似性,以阐明ＣＮＴＦ对于造血系统的可能作用。     

应用微生物代谢调控理论的进展

本文从现代分子生物学的观点,结合酒精、氦基酸发酵等工业生产实际,介绍了在微生物细胞内基因对酶合成的调节(包括底物、类底物的诱导及产物的反馈阻遏或脱阻遇),寡聚酶亚基的解离与聚合、共价修饰以及终产物或中间产物对酶活性的影响,细胞膜透性对代谢调节的影响等现代代谢调节的基本内容,并从理论上论述了发酵生产中提高产量的方法。也介绍了当前国际上引人注目的新动向:固定化细胞及基因工程技术动态及其在发酵工业中应用动态。     

Cloning, expression and reactivating characterization of glycerol dehydratase reactivation factor from Klebsiella pneumoniae XJPD-Li

Genes dhaF and dhaG encoding the α and β subunits of glycerol dehydratase reactivation factor (GDHtR) were amplified from the genomic DNA of Klebsiella pneumoniae XJPD-Li. The identity of the deduced amino acid sequence of the β subunit was relatively low compared with that of K. pneumoniae (U30903), where the 96th amino acid residue was found to be the more active amino acid histidine instead of glutamine in K. pneumoniae (U30903). A specific GDHtR activity of approximately 30 U/mg was attained in Escherichia coli BL21 (pET-28a (+)-dhaFG). His6-tagged GDHtR was purified by Ni-nitrilotriacetate chromatography, and the enzyme was purified 2.6-fold in a yield of 20.7%. The study showed that both glycerol and O₂-inactivated glycerol dehydratase (GDHt) could be quickly reactivated by GDHtR in the presence of ATP, Mg²⁺ and coenzyme B₁₂. However, the glycerol-inactivated GDHt was more easily reactivated than O₂-inactivated GDHt. In the first 10 min of the reactivation reaction, the average reactivation rate was 0.18 and 0.12 μmol/min for glycerol and O₂-inactivated GDHt, respectively.     

Recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li: induction optimization, purification and characterization

Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3-propanediol from glycerol. The optimization of inducting process for recombinant GDHt from Klebsiella pneumoniae XJPD-Li carried out to increase specific activity and ratio of soluble form. The optimum condition was inducing under the isopropyl-beta-D-thiogalactoside concentration of 0.8 mM and the temperature of 20 degrees C for 3 h. Homogeneity of GDHt then was obtained by affinity chromatography, resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of GDHt were pH 8.0 and 45 degrees C, respectively. The K(m) for glycerol, 1,2-propanediol, 1,2-ethanediol and coenzyme B12 were 0.48, 1.43, 3.07 mM, and 10.03 nM, respectively. The GDHt showed relatively stable even under temperature of 40 degrees C and a bit blunt to oxygen. The thermo-inactivation kinetic models were fit linear under different temperatures.     

Recombinant glycerol dehydratase from <Emphasis Type="Italic">Klebsiella pneumonia</Emphasis> XJPD-Li: induction optimization,purification and characterization

Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3-propanediol from glycerol. The optimization of inducting process for recombinant GDHt from Klebsiella pneumoniae XJPD-Li carried out to increase specific activity and ratio of soluble form. The optimum condition was inducing under the isopropyl-β-D-thiogalactoside concentration of 0.8 mM and the temperature of 20°C for 3 h. Homogeneity of GDHt then was obtained by affinity chromatography, resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of GDHt were pH 8.0 and 45°C, respectively. The K _m for glycerol, 1,2-propanediol, 1,2-ethanediol and coenzyme B12 were 0.48 mM, 1.43 mM, 3.07 mM, and 10.03 nM, respectively. The GDHt showed relatively stable even under temperature of 40°C and a bit blunt to oxygen. The thermo-inactivation kinetic models were fit linear under different temperatures.     

Enhancement of pH stability and activity of glycerol dehydratase from Klebsiella pneumoniae by rational design

Glycerol dehydratase (GDHt) is a key and rate-limiting enzyme in the pathway of 1,3-propanediol (1,3-PD) synthesis. The improvement of GDHt’s stability and enzymatic activity is desirable for the biosynthesis of 1,3-PD. The gldABC gene encoding GDHt of Klebsiella pneumoniae was cloned and expressed in Escherichia coli XL10-Gold, and the mutation sites of GDHt were obtained through prediction by PoPMuSiC program. Consequently, two mutants (KpG60 and KpG525) were developed by rational design through site-mutagenesis based on 3D structure which was constructed from homology modeling. Analyses of enzymatic properties showed that pH stability of the mutants was about 1.25–2 times higher than that of the wild type, and specific activity, V_max and K_cat/K_m of KpG525 were about 1.5–2 times higher than those of the wild type. This work presented a simple and useful measure to improve the performance of industrial enzyme.     

Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions

Journal of Industrial Microbiology & Biotechnology - Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol...     

克雷伯氏肺炎杆菌HR526快速合成1,3-丙二醇发酵特性研究

研究了实验室筛选的一株高产1,3-丙二醇(PDO)菌株克雷伯氏肺炎杆菌HR526(Klebsiella pneumoniae HR526), 在5 L B. Braun发酵罐进行甘油补料流加发酵30 h, PDO达到91.47 g/L, 胞外代谢通量分析显示, PDO在对数中期通量达到最大, 而乳酸在稳定期通量达到最大。结合酶学检测分析了PDO合成关键酶PDO氧化还原酶(PDOR)、甘油脱水酶(GDHt)和甘油脱氢酶(GDH)酶活的变化, PDO氧化还原酶活性在对数中期达到最高, 甘油脱水酶/甘油脱氢酶在对数期远大于稳定期、衰退期, 与代谢通量变化一致甘油脱水酶/甘油脱氢酶活性比例不均衡是3-HPA对数期积累的原因, PDO合成主要集中在对数期, 是生长偶联的代谢产物。     

Molecular Cloning, Co-expression, and Characterization of Glycerol Dehydratase and 1,3-Propanediol Dehydrogenase from Citrobacter freundii

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.     

CAMP-dependent protein kinase: prototype for a family of enzymes

Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The crystal structure of the C subunit also is being solved. The C subunit is maintained in its inactive state by forming a holoenzyme complex with an inhibitory regulatory (R) subunit. This R subunit has a well-defined domain structure that includes two tandem cAMP binding domains at the carboxy-terminus, each of which is homologous to the catabolite gene activator protein in Escherichia coli. Affinity labeling with 8N3 cAMP has identified residues that are in close proximity to the cAMP binding sites and is consistent with models of the cAMP binding sites based on the coordinates of the CAP crystal structure. An expression vector was constructed for the RI subunit and several mutations have been introduced. These mutations address 1) the major site of photoaffinity labeling, 2) a conserved arginine in the cAMP binding site, and 3) the consequences of deleting the entire second cAMP binding domain.     

Human mitochondrial DNA polymerase holoenzyme: reconstitution and characterization

We have reconstituted the holoenzyme of the human mitochondrial DNA polymerase from cloned and overexpressed catalytic and accessory subunits. We have examined the polymerization activity of the catalytic subunit alone and of the holoenzyme to establish the function of the accessory subunit in this two subunit enzyme. The accessory subunit associates with the catalytic subunit with a dissociation constant of 35 +/- 16 nM as measured by the concentration dependence of its effect in stimulating maximal DNA binding and polymerization. At saturating concentrations, the accessory subunit contributes to every kinetic parameter examined to facilitate tighter binding of DNA and nucleotide and faster replication. The accessory protein makes the DNA binding 3.5-fold tighter (K(d) of 9.9 +/- 2.1 nM compared to 39 +/- 10 nM for the catalytic subunit alone) without significantly affecting the DNA dissociation rate (0.02 +/- 0.001 compared to 0.03 +/- 0.001 s(-)(1)). The ground-state nucleotide binding is improved from 4.7 +/- 2.0 to 0.78 +/- 0.065 microM, and the maximum DNA polymerization rate is increased from 8.7 +/- 1.1 to 45 +/- 1 s(-)(1) by the addition of the accessory protein. This leads to an increase in processivity from an estimated 290 +/- 46 to 2250 +/- 162. Although the accessory protein has been described as a "processivity factor" because of its effect on the ratio of rate constants defining processivity, this terminology falls short of adequately describing the profound effects of the small subunit on nucleotide-binding and incorporation catalyzed by the large subunit. By using the complete holoenzyme, we can now proceed with a comprehensive analysis of the structural and mechanistic determinants of enzyme specificity that govern toxicity of nucleoside analogues used in the treatment of viral infections such as AIDS.     

产1,3-丙二醇菌株的诱变和筛选

为提高克雷伯氏肺炎杆菌产1,3-丙二醇的能力,以离子束、紫外线和氯化锂为复合诱变法,建立了产酸圈和产物耐受相结合的平板筛选方法,获得可耐受高浓度1,3-丙二醇并且副产物中乙醇含量较少的优良突变菌株2株。与出发菌株相比,两株高产突变菌株Klebsiella pneumoniae LM 03和Klebsiella pneumoniae LM05的1,3-丙二醇产量分别提高了33% 和30% ,达到66.74 g/L和65.12 g/L;乙醇产量分别降低了38% 和24% ,降低为6.59 g/L和8.05 g/L。同时测定了诱变前后还原途径中甘油脱水酶（GDHt）和1,3-丙二醇氧化还原酶（PDOR）的酶活变化,研究表明诱变对GDHt有明显的促进作用,而对PDOR的影响不明显。该诱变和筛选方法目标明确、易操作、效率高,在1,3-PD工业规模的生物法生产中将具有良好的应用价值,而且对于其他具有工业应用价值的菌株筛选工作也具有一定的借鉴意义。