Inactivation of human glutamate dehydrogenase by aluminum

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k_inact of 2.7 min^-1 and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP⁺, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 M. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD⁺-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in helices and sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.Received 25 July 2003; received after revision 27 August 2003; accepted 15 September 2003     

固定化原生质体产谷氨酸脱氢酶的研究

本文介绍了用海藻酸钙凝胶固定化谷氨酸棒杆菌T_(6-13)原生质体生产谷氨酸脱氢酶GDH,Eel.4.1.4的研究。固定化原生质体培养72h后发酵液中GDH活力可达到1.64×~(-2)U/mL,为游离细胞内产酶的205%.固定化原生质体还可用溶菌酶处理固定化细胞而制得,与直接制得的固定化原生质体具有同样的产酶能力,且制备方便,可重复使用,具有良好的贮藏稳定性。     

谷氨酸、生物节律与运动

生物节律是指生物在自然选择、长期进化过程中保存下来的适应性表象。谷氨酸(Glu)是中枢神经系统(CNS)中主要的兴奋性氨基酸类神经递质,已经证实谷氨酸含量有近似昼夜节律现象,同时谷氨酸在调节人体生物节律的过程中扮演重要角色。最近的研究发现,择时运动对谷氨酸的含量及近似昼夜节律会产生明显影响,对运动队异地训练和比赛时快速地调整时差、延缓疲劳有重要意义。     

谷氨酸棒杆菌发酵生产1,5–戊二胺的代谢流分析

为了提高糖类的利用效率,加强糖类代谢向生成尸胺的方向流动,提高尸胺产量,对谷氨酸棒杆菌合成尸胺的代谢网络进行分析,找出影响尸胺合成的代谢流量分配规律和关键节点,并通过改变溶氧及添加辅酶NADPH对关键节点进行验证.结果表明:6–磷酸葡萄糖和丙酮酸是影响碳源流向尸胺合成的关键节点,增强磷酸戊糖途径(HMP)和三羧酸循环(TCA)可以弱化由6–磷酸葡萄糖生成6–磷酸果糖和由丙酮酸生成乳酸,促进碳源向合成尸胺的方向流动,从而有效地提高尸胺产量(产量增幅为77.8%).该研究为高产尸胺的谷氨酸棒杆菌工程菌种改造以及发酵控制提供了理论基础.     

类产碱假单胞菌谷氨酸脱氢酶酶学性质的研究

类产碱假单胞菌的谷氨酸脱氢酶水平受氮源调节,以氮为唯一氮源时酶水平有明显提高,研究表明,该酶的最适反应温度为５０℃,催化氢化反应和脱氨反应的最适ｐＨ值分别为８．０和８．８,金属离子及嘌呤核苷酸对酶活力的影响不显著。     

新生期注射谷氨酸单钠对大鼠脑区损伤程度的比较观察

比较观察了在新生期腹腔内注射不同剂量谷氨酸单钠后,成年大鼠16个脑区的神经元损伤程度．发现大多数脑区的神经元显著减少,但有的脑区对谷氨酸单钠的神经毒性具有一定保护作用．     

Na+ mechanism of δ-opioid receptor induced protection from anoxic K+ leakage in the cortex

Activation of δ-opioid receptors (DOR) attenuates anoxic K⁺ leakage and protects cortical neurons from anoxic insults by inhibiting Na⁺ influx. It is unknown, however, which pathway(s) that mediates the Na⁺ influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na⁺ influxes mediated by voltage-gated Na⁺ channels; (2) DOR activation inhibited Na⁺ influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K⁺ derangement; and (3) DOR activation had little effect on Na⁺/Ca²⁺ exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K⁺ derangement by restricting Na⁺ influx mediated by Na⁺ channels and NMDA receptors, and that non-NMDA receptors and Na⁺/Ca²⁺ exchangers, although involved in anoxic K⁺ derangement in certain degrees, are less likely the targets of DOR signal. Received 26 November 2008; received after revision 26 December 2008; accepted 13 January 2009     

Mechanisms of synaptic depression triggered by metabotropic glutamate receptors

Glutamate, by activation of metabotropic receptors (mGluRs), can lead to a reduction of synaptic efficacy at many synapses. These forms of synaptic plasticity are referred to as long-term depression (mGluR-LTD). We will distinguish between mGluR-LTD induced by pre- or postsynaptic receptors and mGluR-LTD induced by the locus of the expression mechanism of the synaptic depression. We will also review recent evidence that mGluR-mediated responses themselves are subject to depression, which may constitute a form of metaplasticity. Received 13 May 2008; received after revision 07 July 2008; accepted 11 July 2008     

瓜氨酸生产相关基因簇在谷氨酸棒杆菌中的组成型表达

构建1种组成型载体并将载体应用在表达瓜氨酸相关基因簇argCJBDF上。通过去除pXMJ19诱导型启动子上游阻遏蛋白lacI基因的方法,构建组成型质粒pXMJ19-lacI,并将谷氨酸棒杆菌中合成瓜氨酸途径的基因簇argCJBDF克隆到改造过的组成型载体中,实现瓜氨酸合成相关基因簇argCJBDF在谷氨酸棒杆菌的组成型表达。结果表明:重组菌在30℃摇瓶发酵72 h后,N-乙酰谷氨酸激酶的酶活达到(0.323±0.015)U/mg,瓜氨酸的产量达到4.33 g/L。成功构建的组成型表达载体,实现了外源基因簇argCJBDF在谷氨酸棒杆菌中的组成型表达。