停流时间分辨光谱研究NADH-辣根过氧化物酶体系的反应动力学

NADH因其在生物体内氧化-还原过程中的重要作用而引起了广泛的兴趣.NADH可被氧化成NAD+,细胞中NADH/NAD+的比率直接影响细胞的衰老、死亡、癌变.目前还未见利用停流时间分辨光谱法研究该反应的报道,在此研究溶解氧存在的情况下,HRP催化NADH氧化反应的动力学行为.     

从头算分析NAD+及NADP+的电子传递

用HF从头算计算NAD+及NADP+及其还原产物NADH及NADPH分子的绝对能量、电荷分布、前线轨道能量、偶级距、键长、电子自旋伸展空间,并讨论了空间构形与电子传递的关系,提出了NAD+及NADP+在电极和生物氧化过程中传递电子的机理.     

SIOC-AA-005的抗肿瘤作用机制研究

探讨SIOC-AA-005抗肿瘤的作用机制.采用测定HT-29 细胞线粒体膜流动性、线粒体ATP含量、线粒体ATP酶活性及NADH-CoQ氧化还原酶活性、细胞质膜NADH氧化酶活性的方法,对其作用机制进行了深入探讨;SIOC-AA-005在10 nmol/L剂量下可明显降低线粒体膜的流动性,并使线粒体膜电位显著升高;在此浓度下,SIOC-AA-005还可使HT-29细胞内的ATP耗竭,ATP酶活性受到抑制,线粒体中能量产生过程受阻.在胞外反应体系中,SIOC-AA-005对NADH-CoQ氧化还原酶有强烈的抑制作用,对细胞质膜NADH氧化酶也具有较强的抑制作用.SIOC-AA-005可能是通过抑制线粒体NADH-CoQ氧化还原酶、抑制细胞质膜NADH氧化酶以及线粒体ATP酶活性,从而使线粒体膜流动性下降,膜电位升高,细胞内ATP耗竭,最终使肿瘤细胞死亡。     

氧对玉米根细胞质膜氧化还原活性及吡啶核苷酸含量的影响

玉米(Zea mays)根尖的氧化还原活性随介质环境氧的浓度增高而升高,当氧分压达17.262 kPa时,氧化还原活性趋于平稳。有氧与缺氧的根尖各种辅酶的含量有显著差异,其中NADPH和NAPP~+的含量与氧化还原活性的变化呈正相关,但与NADH和NAD~+的含量关系不大。缺氧根再供氧,其NADPH和NADP~+含量随加氧的时间而升高,而NADH和NAD~+的含量则相应减少。供氧根比缺氧根NAD激酶活性提高约20％,表明NAD激酶在有氧条件下与辅酶Ⅰ转换为辅酶Ⅱ有关。     

大鼠再生肝8种肝脏细胞的生物氧化相关基因转录谱预示的生理活动

为了解大鼠肝再生中8种肝脏细胞的生物氧化相关基因转录谱及其预示的生理活动,用Percoll密度梯度离心结合免疫磁珠分选方法分离大鼠再生肝的肝细胞、胆管上皮细胞、卵圆细胞、星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞和树突状细胞等8种细胞,用Rat Genome2302.0芯片等检测生物氧化相关基因在上述细胞中表达变化,用H-Cluster等软件及生物信息学和系统学生物等方法分析它们的表达模式及预示的生理活动.结果表明:38个生物氧化相关基因在大鼠肝再生中发生了有意义表达变化,相应细胞的基因数为6,32,0,2,3,1,3,2个.肝再生启动阶段和进展阶段的NAD+合成增强,从NADH到O2的电子传递及ATP合成增强.终止阶段的FADH2分解减弱,从FADH2到O2的电子传递减弱.结论:大鼠肝再生与生物氧化密切相关.     

Effects of Iron on Hydrogen-producing Capacity,Hydrogenase and NADH-fd Reductase Activities of a Fermentative Hydrogen-producing Bacterial Strain B49

0　IntroductionHydrogenisaclean ,effectiveandrenewableener gy ,andisanultimatereplacementforfossilhydrocarbonfuelsinthefuture .Biohydrogenproductionfromwasteisattractiveduetoenergyrecoveryandenvironmentalcleanupatthesametime .Biohydrogengenerationbypho to andnon photosyntheticbacteriahasbeenstudiedex tensively[1 4 ] .Ingeneral ,thesemicroorganismscontainFe Sproteins .Forexample ,inhydrogen producingClostridiumcellulolyticumtherearehydrogenase ,NADH fdreductase ,fd NADP+reductase ,fd NAD+…     

高产光学纯(R)-乙偶姻工程菌株的构建与发酵工艺优化

本研究拟在大肠杆菌中构建光学纯(R)-乙偶姻的合成途径和利用辅酶工程调控NADH/NAD+氧化还原平衡,并对工程菌发酵(R)-乙偶姻进行优化。将来源于Enterobacter cloacae的α-乙酰乳酸合成酶基因budB、α-乙酰乳酸脱羧酶基因budA和来源于Lactobacillus brevis的NADH氧化酶基因noxE进行密码子优化后组成基因簇,构建表达质粒并导入大肠杆菌,进一步优化工程菌的培养基成分和发酵条件,提高(R)-乙偶姻的合成能力。结果表明:获得专一性合成高光学纯(R)-乙偶姻的大肠杆菌工程菌株GXASR,对其发酵条件进行系统优化后,摇瓶发酵的(R)-乙偶姻产量为36.82g/L,光学纯度达99.1%,发酵罐补料发酵的(R)-乙偶姻产量达到67.65g/L。在大肠杆菌细胞中过表达外源基因簇budB-budA-noxE能够高效合成光学纯(R)-乙偶姻,经发酵优化后,工程菌的(R)-乙偶姻产量、生产强度和得率均显著提高,为代谢工程改造大肠杆菌生产高光学纯(R)-乙偶姻提供了理论基础。     

植物蛋白乙酰化/去乙酰化与代谢和基因表达调控研究进展

植物细胞中组蛋白赖氨酸乙酰化修饰对基因表达具有重要的作用,同时影响糖酵解、三羧酸循环和光合作用途径中非组蛋白的代谢酶活性。因此,识别参与调控代谢酶活性的HAT和HDAC具有重要意义。相反,代谢物如乙酰-Co A和NAD+参与蛋白可逆乙酰化进程,并且它们在细胞内的水平可能影响组蛋白乙酰化酶和组蛋白去乙酰化酶的活性。从组蛋白质赖氨酸乙酰化可逆过程、蛋白乙酰化酶和组蛋白去乙酰化酶分类及其细胞内分布和代谢酶的活性研究阐述了HAT和HDAC的作用及其研究进展。     

An electrochemical and Raman spectroscopic study on involvement of La3+ ions in lactate dehydrogenase catalysis

The influence of La 3+ on lactate dehydrogenase catalysis was investigated by electrochemical and FT-Raman techniques. The results showed that La 3+ ions bind preferably with phosphate moiety and ribose group (3′-OH) connected to nicotinamide in coenzyme NAD +/NADH, destabilize the enzyme-coenzyme complex and inhibit LDH activity.     

线粒体呼吸链抑制剂对青蒿素代谢速率的影响

线粒体呼吸链在青蒿素抑制酵母的过程中起重要作用。为了研究线粒体呼吸链对青蒿素代谢的影响,利用不同的抑制剂抑制线粒体呼吸链,用薄层层析法检测青蒿素代谢速率。结果表明:还原型烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶抑制剂(DPI)可以减缓酵母细胞和疟原虫细胞对青蒿素的代谢,而呼吸链复合物Ⅱ、Ⅲ、Ⅳ的抑制剂对青蒿素消耗没有明显影响。NADH脱氢酶在青蒿素代谢中起重要作用,NADH脱氢酶结构上存在巨大差异。这也为解释青蒿素的物种选择性毒性提供了线索。     

Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I

Hydrogenosomes are double-membraned ATP-producing and hydrogen-producing organelles of diverse anaerobic eukaryotes. In some versions of endosymbiotic theory they are suggested to be homologues of mitochondria, but alternative views suggest they arose from an anaerobic bacterium that was distinct from the mitochondrial endosymbiont. Here we show that the 51-kDa and 24-kDa subunits of the NADH dehydrogenase module in complex I, the first step in the mitochondrial respiratory chain, are active in hydrogenosomes of Trichomonas vaginalis. Like mitochondrial NADH dehydrogenase, the purified Trichomonas enzyme can reduce a variety of electron carriers including ubiquinone, but unlike the mitochondrial enzyme it can also reduce ferredoxin, the electron carrier used for hydrogen production. The presence of NADH dehydrogenase solves the long-standing conundrum of how hydrogenosomes regenerate NAD+ after malate oxidation. Phylogenetic analyses show that the Trichomonas 51-kDa homologue shares common ancestry with the mitochondrial enzyme. Recruitment of complex I subunits into a H2-producing pathway provides evidence that mitochondria and hydrogenosomes are aerobic and anaerobic homologues of the same endosymbiotically derived organelle.     

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (I(e)) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that I(e) is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates I(e), because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.     

酶学信号扩增方法定量检测生物素探针杂交

将靶核酸固定于微孔滴定板上，用化学方法标记的生物素探针进行杂交，采用底物－辅助因子循环信号扩增系统对杂交结果进行检测，结果表明用该信号检测系统的检验灵敏度比常用显色底物的检测灵敏度高１０倍。     

A candidate NAD+ transporter in an intracellular bacterial symbiont related to Chlamydiae

Bacteria living within eukaryotic cells can be essential for the survival or reproduction of the host but in other cases are among the most successful pathogens. Environmental Chlamydiae, including strain UWE25, thrive as obligate intracellular symbionts within protozoa; are recently discovered relatives of major bacterial pathogens of humans; and also infect human cells. Genome analysis of UWE25 predicted that this symbiont is unable to synthesize the universal electron carrier nicotinamide adenine dinucleotide (NAD+). Compensation of limited biosynthetic capacity in intracellular bacteria is usually achieved by import of primary metabolites. Here, we report the identification of a candidate transporter protein from UWE25 that is highly specific for import of NAD+ when synthesized heterologously in Escherichia coli. The discovery of this candidate NAD+/ADP exchanger demonstrates that intact NAD+ molecules can be transported through cytoplasmic membranes. This protein acts together with a newly discovered nucleotide transporter and an ATP/ADP translocase, and allows UWE25 to exploit its host cell by means of a sophisticated metabolic parasitism.     

盐生杜氏藻和衣藻双结构域NAD+-GPD基因的生物信息学分析

盐生杜氏藻（Dunaliella salina）是极端耐盐的单细胞真核绿藻,依赖于NAD的3－磷酸甘油脱氢酶（NAD+-GPD）是盐生杜氏藻调渗物质——甘油合成的关键酶.盐生杜氏藻NAD+-GPD基因是第一个被发现含双结构域（SerB和GPD）的GPD基因.而双结构域可能是盐生杜氏藻具有极强耐盐性和快速合成甘油的关键.通过与莱茵衣藻基因组数据库比对,发现莱茵衣藻（Chlamydomonas reinhardtii）中也存在具有SerB和GPD结构域的NAD+-GPD基因序列.在对两个物种NAD+-GPD基因的基因结构、mRNA组成成分、编码蛋白及其理化性质和编码蛋白结构的分析中,发现二者具有较高的相似性.针对目前仅在盐藻和衣藻中发现含SerB和GPD结构域的NAD+-GPD基因这一现象,分别对SerB和GPD结构域同源基因的系统进化进行了分析.     

氧脱木素过程中羟基自由基的控制

氧脱木素的主要问题是提高其脱木素选择性,而氧脱木素过程中产生的含氧自由基是影响其脱木素选择性的主要因素.而本文用改进的Fenton反应Co2 -H2O2体系产生羟基自由基(HO.),用茜素紫3B的氧化来显色测定HO..将Co2 -H2O2体系与深度脱木素竹浆进行反应,并通过添加HO.捕获剂来控制HO.的数量,研究HO.与竹浆中木素和碳水化合物的反应情况.把HO.捕获剂作为助剂用于深度脱木素竹浆氧脱木素过程,研究HO.的量对氧脱木素过程的影响.研究结果表明,HO.能与木素和碳水化合物发生反应,具有脱木素作用,但对碳水化合物的降解也较严重.HO.捕获剂具有清除HO.的作用,在氧脱木素过程中加入HO.捕获剂能减少碳水化合物的降解,提高氧脱木素选择性.     

X-ray study of the lithium complex of NAD.

The Li+-NAD+ complex exists as a 'dimer' of two molecules arranged head-to-tail with Li+ coordinated tetrahedrally to adenine N(7) and three pyrophosphate oxygens. Adenine is stacked intermolecularly on nicotinamide. The conformation of NAD+ is 'extended' and similar to that found in holoenzyme complexes. This is in contrast to the 'folded' structure proposed from spectroscopic studies.     

Identification of the BAL-labile factor

One of us has previously reported that treatment of the Keilin and Hartree heart-muscle preparation with 2,3-dimercaptopropanol (BAL), in the presence of air, leads to the complete inactivation of the succinate oxidase system with little if any effect on the activities of succinate dehydrogenase (until more than half the BAL was oxidized) or cytochrome c oxidase. The inactivation of the complete succinate oxidase system requires the oxidation of BAL by air in the presence of the enzyme. It is not caused by H2O2 or BAL disulphides produced during the oxidation of BAL. Spectroscopic studies identified the block as lying between cytochromes b and c. It was suggested that a BAL-labile factor is present which transfers electrons from cytochrome b to cytochrome c and which is destroyed by coupled oxidation with BAL. The factor is also required for NADH oxidation. Subsequent work showed it is not identical with cytochrome c1 (ref. 4), myoglobin present in the preparation or the antimycin-binding site. We report here that this factor is identical to the iron-sulphur protein in the central portion of the respiratory chain first identified by Rieske.     

YBCO超导体中含氧量的测定

ＹＢＣＯ超导体中含氧量和它的超导性有很大关系。本文用氢溴酸溶样，加柠檬酸盐与ＣＵ２＋生成配合物，然后用碘量法测含氧量，取得较好结果．