甘氨酸神经递质研究进展

甘氨酸是化学结构最简单的氨基酸,但具有复杂的功能。甘氨酸在中枢神经系统中是介导快速抑制性神经传递的一种重要的神经递质,在控制神经元兴奋性方面发挥重要作用。就其神经递质功能对甘氨酸的生物合成、释放与调控以及作用模式等方面的近年研究进展做一综述,对甘氨酸神经递质的全面认识将有益于炎性痛、痉挛状态以及癫痫等中枢神经系统疾病的诊断、预防及治疗。     

甘氨酸合成工艺

介绍了一种新的甘氨酸合成方法。与国内现行工艺相比,产品收率从７０％提高８２％,并对各影响因素作了讨论,通过对反应的研究,证明这是一种收率高、成本低、三废少、有工业化价值的方法。     

甘氨酸受体的研究进展

甘氨酸受体(GlyR)是中枢神经系统中一种重要的抑制性受体.GlyR是氯离子(Cl^－)选择性通道蛋白,属于配体门控离子通道超家族的一员.天然GlyR是由α和β亚基组装而成的五聚体.介绍了近年来有关GlyR的结构、功能、药理特性研究的重要进展,并结合本实验室工作,论述GlyR的调制及其可能机制.     

甘氨酸生产新工艺中Cl~-含量测定方法的研究

采用不同的氯离子(Cl-)含量测试方法(摩尔法、氯离子选择电极法、电位滴定法、电导法、比浊法),对甘氨酸合成中各步骤的产物分别进行Cl-含量测试,确定出相适应的测定方法。     

甘氨酸受体在肾细胞缺氧保护中的作用研究

目的明确甘氨酸受体(glycine receptor,GlyR)是否介导甘氨酸对ATP耗竭细胞的保护作用。方法构建甘氨酸受体α1亚单位(GlyRα1)的真核表达载体pcDNA3.1(b),转染入缺乏天然GlyR的人胚胎肾细胞HEK293。化学性缺氧使细胞处于ATP耗竭状态,光学显微镜及电镜观察细胞形态及超微结构改变,LDH释放率、细胞膜对荧光标记复合物通透性反映细胞膜完整性,台盼蓝染色观察细胞活性。结果细胞ATP耗竭3h后,细胞膜通透性明显增加,细胞器结构损伤,大量细胞死亡。甘氨酸明显降低表达GlyR的转染HEK293细胞膜通透性,阻止荧光标记复合物进入细胞内,细胞LDH释放率由59.18±8.10%下降为35.15±2.61%。从而维持细胞形态结构,降低细胞死亡率。对不表达GlyR细胞无保护作用。结论GlyR介导甘氨酸对ATP耗竭细胞的保护作用,增强细胞对ATP耗竭的耐受能力,增加细胞的存活几率。     

中枢甘氨酸受体分子特性的研究

目录一、ＧｌｙＲ在ＣＮＳ中的分布二、ＧｌｙＲ的分子结构　 (一 )亚单位结构　 (二 )受体组装和表达　 (三 )配体结合　 (四 )通道孔区　 (五 )通道激活　 (六 )磷酸化三、ＧｌｙＲ分子病　 (一 )家族性惊厥病　 (二 )鼠惊厥综合病　 (三 )肌阵挛　 (四 )遗传性惊厥病的生理趋同现象四、结语甘氨酸受体 (ＧｌｙＲ)介导的抑制性神经传递在哺乳动物中枢神经系统 (ＣＮＳ)反射活动、随意运动调节和感觉信号的处理中具有重要作用[1] 。ＧｌｙＲ五聚体由三个独立的多肽组成 :两个糖蛋白 (48ｋＤ和 5 8ｋＤ) ,分别称为α和 β亚单位 ,另一个为 9…     

在银溶胶中甘氨酸和甘氨酸二肽的表面增强拉曼散射

甘氨酸及Gly-Gly和Gly-L-Pro二肽的表面增强拉曼散射谱与溶液内的普通拉曼谱的对比研究表明,银胶对COO、NH_2、C-N和C-C基团的振动有明显的增强.对CH_2基团无增强.Gly-Gly的SERS与pH有关,接近中性pH值时增强效果好,在酸性和碱性溶液内效果差.在 Gly-L-Pro的SERS谱中有强的酰胺Ⅰ带,而 Gly-Gly则几乎观察不到.     

甘氨酸及其衍生物的抗菌作用

本文讨论甘氨酸、甘氨酸盐、甘氨酸高级脂肪醇的酯以及其它甘氨酸衍生物的抗菌作用。     

Purification and properties of a novel glycine amino peptidase from Actinomucor elegans and its potential application

AIMS: To study the properties and show the potential application of a glycine aminopeptidase from Actinomucor elegans. METHODS AND RESULTS: The enzyme was estimated to have molecular mass of 320 kDa by gel filtration and the subunit size of 56.5 kDa by SDS-PAGE. It hydrolysed glycine from substrate more efficiently than other amino acids. The optimal temperature for this enzyme was 40 degrees C and at pH 8.0 it showed its highest activity. The Km and Kcat of the enzyme for glycine-beta-naphthylamine was 0.24 mm and 100.8 s(-1), respectively. Zinc, copper, cadmium and o-phenanthrolin suppressed almost all enzyme activities at the concentration of 1.0 mm. In the process of hydrolysing proteins, it could improve the protease activity considerably. CONCLUSIONS: It was a hexamer metalloenzyme which was specific for the substrates with glycinse residue at the N-terminal and some metal cations were needed to maintain its activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the properties of a novel aminopeptidase and shows its potential application in the process of the food industry.     

Novel aminopeptidase specific for glycine from Actinomucor elegans

Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3% of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly > Ala > Met > Arg > Ser > Leu. The Km value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40 degrees C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.     

Purification and Properties of a Type II-like Dipeptidyl Peptidase from the Ruminal Peptidolytic Bacterium,Prevotella albensis M384

A dipeptidyl peptidase which hydrolyses the synthetic dipeptidyl peptidase (DPP) substrate, Ala₂- p -nitroanilide, was purified 193-fold from the ruminal peptidolytic bacterium, Prevotella albensis M384. The enzyme was a homodimer of molecular mass 91 kDa. Its activity against Ala₂- p -nitroanilide had optimal pH and temperature of 7.2 and 40°C respectively. Enzyme activity was inhibited by the serine protease inhibitors, PMSF and dichloroisocoumarin, but not by inhibitors of other categories of proteases. Synthetic substrates for DPP-1 (GlyArg- p -nitroanilide, GlyArg-4-methoxy-naphthylamide), DPP-3 (ArgArg-4-methoxynaphthylamide) and DPP-4 (GlyPro-4-methoxynaphthylamide) or for leucine or alanine aminopeptidase were not hydrolysed, nor were di- or tripeptides. N-Acetyl-Ala₂- p -nitroanilide was not hydrolysed. Oligopeptides with Ala, Ile, Ser or Val adjacent to the N-terminal amino acid were all hydrolysed, while peptides with basic or acidic residues in the same position were not. The purified DPP from P. albensis is therefore most similar in its catalytic properties to mammalian DPP-2.     

Purification and Characterization of a Basic Amino Acid-Specific Peptidase from Seeds of Jack Bean (Canavalia ensiformis)

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60°C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.     

Pharmacological Properties of Glycine Transport in the Frog Retina

The high-affinity glycine transport in neurons and glial cells is the primary means for inactivating synaptic glycine. Two different glycine transporter genes, Glyt-1 and Glyt-2, have been cloned. Glyt-1 has been reported to occur in the retina, but there is no evidence for expression of the Glyt-2 transporter. We have pharmacologically characterized glycine transport in the frog retina. 3H-Glycine uptake in the retina was insensitive to modulation by phorbol esters or changes in cAMP levels, and was partially inhibited by sarcosine. Differential sensitivity of glycine transport to sarcosine was exhibited by synaptosomal fractions from the inner and outer plexiform layers of the frog retina. The Na+ Hill coefficient of glycine uptake was 2.0, as has been reported for Glyt-2. In addition, amoxapine, a specific inhibitor of the Glyt-2a isoform, reduced by 60% glycine uptake by P2 synaptosomal fraction. Our results indicate the presence of different glycine transporter isoforms in the frog retina, acting mainly through the classical inhibitory glycine system.     

Peptidase N of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa possesses a peptidase N activity analogous to those described in Escherichia coli and Salmonella typhimurium . This activity resides in a protein with an M _r value of 85 000. Part of this peptidase activity appears to be associated with the cytoplasmic membrane. The K _M value for this peptidase bound to the cytoplasmic membrane is in the range of 0.5 mM.     

固定甘氨酸用于吸附水中金属离子的研究

采用环氧氯丙烷法将惰性载体Sephadex G-25活化,使其与甘氨酸偶联从而得到固定羧基的离子交换吸附剂。对该吸附剂吸附金属离子性能的研究表明,在pH 9.0时,吸附剂对Ca2+等金属离子有很强的吸附,且对Fe2+、Fe3+、Mn2+的吸附力比对Ca2+和Mg2+的吸附力要强。16g(湿重)吸附剂对金属离子的饱和吸附量分别为:Ca2+16.99mg,Mg2+6.86mg,Fe2+10.06mg,Fe3+4.93mg,Mn2+11.51mg。同时,该吸附剂具有稳定性好、能重复使用且制备成本低等特点,在污水处理、金属离子回收等方面有很好的应用前景。     

Characterization of two M17 family members in Escherichia coli, Peptidase A and Peptidase B

Escherichia coli encodes two aminopeptidases belonging to the M17 family: Peptidase A (PepA) and Peptidase B (PepB). To gain insights into their substrate specificities, PepA or PepB were overexpressed in ΔpepN, which shows greatly reduced activity against the majority of amino acid substrates. Overexpression of PepA or PepB increases catalytic activity of several aminopeptidase substrates and partially rescues growth of ΔpepN during nutritional downshift and high temperature stress. Purified PepA and PepB display broad substrate specificity and Leu, Lys, Met and Gly are preferred substrates. However, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and insulin B chain peptide. Importantly, this strategy, i.e. overexpression of peptidases in ΔpepN and screening a panel of substrates for cleavage, can be used to rapidly identify peptidases with novel substrate specificities encoded in genomes of different organisms.     

Characterization and role of Peptidase N from Salmonella enterica serovar Typhimurium

ATP-independent peptidases are important during the distal steps of cytosolic protein degradation. The contribution of a member of this group, Peptidase N (PepN) was studied in Salmonella enterica serovar Typhimurium (Salmonella typhimurium). The DeltapepN strain displays greatly reduced cleavage of 9 out of a total of 13 exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. The cleavage profile of purified S. typhimurium PepN is Arg>Ala>Thr, demonstrating broad specificity. Comparative biochemical studies with purified PepN from Escherichia coli and S. typhimurium revealed the latter to be distinct: S. typhimurium PepN cleaves Thr-AMC more efficiently and is less sensitive to inhibition by N-ethylmaleimide. Studies with DeltapepN and PepN overexpression demonstrated its importance for growth during nutritional downshift in combination with high temperature stress. In summary, S. typhimurium PepN contributes significantly to cytosolic aminopeptidase activity and its role is manifested under selected stress conditions.     

雅致放射毛霉AS3.2778碱性蛋白酶的纯化及性质研究

利用盐析,离子交换,疏水层析及凝胶过滤的方法从雅致放射毛霉AS3.2778的发酵麸曲中分离纯化出一碱性蛋白酶,其纯化提高了22.7倍,酶活回收率16.1%,最终比酶活可达到6094u/mg。电泳分析发现,该蛋白酶是一单体蛋白,其分子量大约在32KDa。性质分析表明：该蛋白酶在60℃、pH8.5～10.5具有最大催化活性;在40℃以下,pH6.0～9.0的范围有很好的稳定性;1mM的PMSF可以完全抑制其活性,显示该蛋白酶属于丝氨酸蛋白酶家族。底物专一性的研究发现,该蛋白酶有相当广泛的肽键选择性,对绝大多数由疏水性氨基酸（尤其是亮氨酸）构成的肽键有很强的水解能力。