烟酰胺腺嘌呤二核苷酸和Sirtuins在衰老和疾病中的作用

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD⁺)作为氧化还原反应的重要辅酶,是能量代谢的核心。NAD⁺也是非氧化还原NAD⁺依赖性酶的共底物,包括沉默信息调节因子(Sirtuins)、聚ADP-核糖聚合酶(poly ADP-ribose polymerases, PARPs)、CD38/CD157胞外酶等。NAD⁺已成为细胞信号转导和细胞存活的关键调节剂。最近的研究表明,Sirtuins催化多种NAD⁺依赖性反应,包括去乙酰化、脱酰基化和ADP-核糖基化。Sirtuins催化活性取决于NAD⁺水平的高低。因此,Sirtuins是细胞代谢和氧化还原状态关键传感器。哺乳动物中已经鉴定并表征了7个Sirtuins家族成员(SIRT1-7),其参与炎症、细胞生长、生理节律、能量代谢、神经元功能、应激反应和健康衰老等多种生理过程。本文归纳了NAD⁺的生理浓度及状态、NAD⁺的生物合成途径,并阐述了Sirtuins的生物学功能,重点讨论了SIRT1-7表达及活性与衰老和疾病之间的关系。此外,本文还进一步探讨了NAD⁺和Sirtuins依赖的衰老机制,机体NAD⁺水平随着年龄增长而下降,导致Sirtuins活性下降,尤其是SIRT1、SIRT3和SIRT6,引发细胞核和线粒体功能下降,衰老及老化相关疾病也随之发生。研究表明,NAD⁺前体补充剂能够快速提高机体NAD⁺水平和Sirtuins活性,是一种有效的抗衰老干预措施,为全球老龄化社会带来希望。     

Coenzyme production using immobilized enzymes. I. Preparation, characterization, and laboratory-scale application of an immobilized NAD kinase

NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, EC2.7.1.23) isolated from chicken liver was immobilized on a silica-based support possessing aldehyde functional groups. The highest catalytic activity achieved was 16 U g⁻¹ solid. The optimal pH for the catalytic activity of the immobilized NAD⁺ kinase was pH 7.1–7.3. The apparent optimum temperature for the immobilized enzyme was about 5°C higher than that of the soluble enzyme. There were no significant differences in the K_{m app} values. The immobilization improved the conformational stability of the enzyme. In preliminary experiments, a 95% conversion of NAD⁺ to NADP⁺ was achieved with use of the immobilized NAD⁺ kinase, which preserved its starting activity practically unchanged up to 36 days.     

转录调控因子Rex的功能及调节机制研究进展

转录因子Rex是一种广泛存在于革兰氏阳性菌,能够与NADH或者NAD⁺直接结合响应胞内NADH/NAD⁺的氧化还原传感器,与靶基因的结合可调节细胞内的多种生理代谢。NAD（H）是调节细胞能量代谢的必需辅酶,显示微生物细胞内的氧化还原状态。研究发现Rex的调节活性与细胞内NADH/NAD⁺比率相关。需氧和厌氧菌属中Rex单体和复合物晶体结构的解析揭示了Rex、NADH/NAD⁺和靶基因间的作用关系及调控机制。通过比较分析了不同菌株中Rex单体和复合物的晶体蛋白结构,并揭示了NADH/NAD⁺对Rex调控活性的影响,进一步解析了Rex与碳和能量代谢、厌氧代谢、发酵、生物膜等之间的联系,并展望了Rex的研究和应用方向。     

Intracellular Ca2+ Chelators Prevent Dna Damage And Protect Hepatoma 1Clc7 Cells From Quinone-Induced Cell Killing

Exposure of hepatoma lclc7 cells to 2,3-drniethoxy-1.4-naphthoquinone (DMNQ) resulted in a sustained elevation of cytosolic Ca²⁺. DNA single strand breaks and cell killing. DNA single strand break formation was prevented when cells were preloaded with either of the intracellular Ca²⁺ chelators. Quin 2 or BAPTA, to buffer the increase in cytosolic Ca²⁺ concentration induced by the quinone. DMNQ caused marked NAD⁺ depletion which was prevented when cells were preincubated with 3-aminobenzamide. an inhibitor of nuclear poly-(ADP-ribose)-synthetase activity. or with either of the two Ca²⁺ chelators. However. 3-aminobenzamide did not protect the hepatoma cells from loss of viability. Our results indicate that quinone-induced DNA damage. NAD⁺ depletion and cell killing are mediated by a sustained elevation of cytosolic Ca²⁺     

Regulation of dinitrogen fixation in intact Azotobacter vinelandii

1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated.

2. The hypothesis that a high (NADH + NADPH)/(NAD⁺ + NADP⁺) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases.

3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.     

辅酶Ⅰ体内代谢调控研究进展

辅酶Ⅰ——烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD⁺）是一种在糖酵解、糖异生、三羧酸循环及呼吸链中发挥重要作用的辅酶,广泛参与DNA修复、组蛋白去乙酰化等生命过程。近年来研究表明NAD⁺合成的前体和中间化合物（具有维生素B3活性的烟酸、烟酰胺、烟酰胺核苷和烟酰胺单核苷酸）在预防糙皮病、延缓衰老,治疗神经和心血管多种疾病、调节胰岛素分泌、调控mRNA的表达等方面具有重要疗效。着重介绍了辅酶Ⅰ体内的合成代谢以及参与的调节衰老进程,以期为利用合成生物学技术在大肠杆菌中富集NAD⁺中间化合物提供理论依据和技术支撑。     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

Menadiol as an electron donor for reversed oxidative phosphorylation in submitochondrial particles

Dithiothreitol in the presence of menadione or N,N,N′,N′-tetramethyl-p-phenylenediamine provides the reducing equivalents for oxidative phosphorylation and the ATP-dependent reduction of NAD⁺ in submitochondrial particles. With menadione the reaction is nearly as fast as with succinate and it is insensitive to antimycin, indicating electron entry between the first and second sites of oxidative phosphorylation. The phenylenediamine-mediated reduction of NAD⁺ is nearly as fast as succinate-linked reduction and is antimycin sensitive.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

A versatile colony assay based on NADH fluorescence

Direct visualization of the activity of enzymes expressed by bacterial colonies attached to a solid support, often referred to as “filter assay”, is a powerful strategy for the identification of new or improved biocatalysts. In this work we demonstrate the usefulness of NAD⁺/NADH coupled enzymatic reactions as visualization tool in such experimental setups. Dehydrogenases, capable of oxidizing or reducing the reaction product released from the bacterial colony were supplemented to the screening solution, together with the screening substrate and a sufficient amount of NAD⁺ or NADH, respectively. We also examined the screening of directly NAD⁺/NADH coupled reactions. The release or consumption of NADH in the area of colonies was monitored on behalf of its fluorescence at 450 nm. Excitation was achieved by standard “black-light” UV tubes (340–360 nm). The visible fluorescence signal was recorded using a CCD-camera. We got excellent results for the screening of threonine aldolases and esterases and were able to show the principle utility for amidase, nitrilase, nitrile hydratase, hydroxynitrile lyase and benzaldehyde dehydrogenase active colonies.     

Ca2+ release induced by cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is the most potent Ca²⁺-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD⁺ by ADP-ribosyl cyclases. cADPR binds to Ca²⁺ channels in the endoplasmic reticulum membrane to activate a Ca²⁺-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca²⁺ concentrations. Thus, cADPR may function as an endogenous regulator of Ca²⁺-induced Ca²⁺ release, and there is excitement that it may also function as a Ca²⁺-mobilizing second messenger.     

核糖核酸5'端NAD+帽子修饰研究进展

m7G帽子具有保护RNA不被降解以及招募相关蛋白参与内含子剪切、poly(A)加尾、出核和翻译等功能。一直以来,它被认为是真核生物mRNA所特有的修饰类型。然而近年来,在包括原核生物在内的多个物种中均检测到一种新的RNA 5’端修饰,即核酸代谢物烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD⁺)帽子。目前NAD⁺修饰RNA(NAD-RNA)的生物学功能研究仍处于起始阶段。本文概述了NAD-RNA的发现及其检测和鉴定技术的发展;探讨了NAD⁺帽子对RNA的调控功能,以及NAD-RNA脱帽和加帽的影响因素;并进一步推测NAD-RNA在生物的生长、发育和环境响应中发挥的潜在功能。最后,展望了未来NAD-RNA的研究方向和主题。     

Rapid microfluorimetry of enzyme reactions in single living cells

An optimized photon counting technique allows the microfluorimetric study of NAD⁺ (or NADP⁺) reduction-reoxidation transients in single living cells with a time resolution in the range of 1/50-1/100 sec. The transients resulting from the micro-electrophoretic addition of metabolites (e.g. Glc-6-P or Glc-1-P) can be analyzed in terms of early parameters (e.g. initial lag, rise half time or full rise time) and overall parameters (time of rise and half decay, amplitude, reoxidation time). Both the initial lag and rise half time are considerably longer with Glc-1-P than with Glc-6-P, possibly due to control at the phosphoglucomutase or compartmentation of glycolytic phosphate esters. While glycolytic NAD⁺ (or NADP⁺) reduction proceeds adequately in aerobic EL2 and EAT ascites cells (although ΔNADH/Δt is higher at anaerobiosis), it is critically dependent upon anaerobiosis in L and astrocytoma cells. Thus by rapid microfluorimetry it is possible to resolve the rising phase or other segments of the fluorescence transients into components each corresponding to a particular step in the sequence of intracellular events or control states.     

Mitochondrial membrane potential, transmembrane difference in the NAD redox potential and the equilibrium of the glutamate-aspartate translocase in the isolated perfused rat heart

The distribution of glutamate and aspartate and the mitochondrial membrane potential (Δψ) were studied in isolated rat heart mitochondria and in the intact perfused rat heart. The diffusion potential imposed by the glutamate-aspartate exchange through mediation of the electrogenic glutamate-aspartate translocator attained a value close to the mitochondrial Δψ measured from the distribution of triphenylmethylphosphonium ion (TPMP⁺) both in isolated mitochondria and in intact myocardium. Distributions of the Δψ probe and metabolites were determined by subcellular fractionation of the heart muscle in a non-aqueous medium. The results indicate that the glutamate-aspartate translocator is in near equilibrium in the myocardium. The diffusion potential of the glutamate-aspartate exchange, and the mitochondrial/cytosolic difference in the redox potentials of the free NAD⁺/NADH pools are equal allowing for experimental error. These data obtained from intact tissue can therefore be interpreted as supporting the notion of the transmembrane uphill transport of reducing equivalent from the cytosolic free NAD⁺/NADH pool being driven by the malate-aspartate cycle energized by the mitochondrial Δψ.     

Pyridine Nucleotide Changes In Hepatocytes Exposed To Quinones

Quinones may be toxic by a number of mechanisms. including arylation and oxidative stress caused by redox cycling. Using isolated hepatocytes, we have studied the cytotoxicity of four quinones. with differing abilities to arylate cellular nucleophiles and redox cycle. in relation to their effects on cellular pyridine nucleotides. High concentrations of menadione (redox cycles and arylates). 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) 2.3-dimethoxy-1.4-naphthoquinone (a pure redox cycler) and p-benzoquinone (a pure arylator) caused an initial decrease in NAD⁺ and loss of viability, which was not prevented by 3-aminobenzamide. an inhibitor of poly(ADP-ribose)polymerase. In contrast. 3-aminobenzamide inhibited the loss of NAD' and viability caused by dimethyl sulphate so implicating poly(ADP-ribose)polymerase in its toxicity but not that of the quinones. Non-toxic concentrations of menadione. 2.3-dimethoxy-1.4-naphthoquinone and 2-hydroxy-1.4-naphthoquinone all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD⁺ was accompanied by a small. transient increase in NADP⁺ and followed by a larger. prolonged increase in NADPH and total NADP⁺ + NADPH. Nucleotide changes were not observed with non-toxic concentrations of p-benzoquinone. Our findings suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides. in an attempt to combat the effects of oxidative stress     

The action of amytal on frog gastric mucosa

The frog gastric mucosa has been shown to be sensitive to amytal. At 2 mM acid secretion was completely inhibited with a rise of resistance, fall in short-circuit current and no significant change in potential difference. Simultaneously there was 75% inhibition of O₂ consumption and 50% depression of ATP levels. Dual-beam spectrophotometric studies of intact mucosa with amytal showed a crossover point between NAD⁺ and FAD. The microsomal NADH oxidase ferricyanide reductase has also been shown to be amytal sensitive. Cl⁻ transport was relatively insensitive to amytal, suggesting a qualitative distinction between the mechanisms underlying the transport of H⁺ and Cl⁻ in the mucosa. This was further brought out by the effects of anoxia in which H⁺ transport was inhibited at 5 min but Cl⁻ transport at minimally 20 min following the onset of anoxia.     

Purification and characterization of leucine dehydrogenase from an alkaliphilic halophile, Natronobacterium magadii MS-3

Leucine dehydrogenase ( -leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) was purified to homogeneity from the crude extract of an alkaliphilic halophile, Natronobacterium magadii MS-3, with a yield of 16%. The enzyme had a molecular mass of about 330 kDa and consisted of six subunits identical in molecular mass (55 kDa). The enzyme required a high concentration of salt for stability and activity. It retained the full activity after heating at 50 °C for 1 h and about 50% activity after being kept at 30 °C for 2 months in the presence of 2.5 M NaCl. The enzyme required NAD⁺ as a coenzyme and showed maximum activity in the presence of more than 3 M salt, as CsCl, RbCl, NaCl, or KCl. In addition to -leucine, -valine and -isoleucine were also good substrates in the oxidative deamination. In the reductive amination, 2-keto analogs of branched-chain amino acids were substrates. The Michaelis constants were 0.69 mM for -leucine, 0.48 mM for NAD⁺, 4.0 mM for 2-ketoisocaproate, 220 mM for ammonia, and 0.02 mM for NADH in the presence of 4 M NaCl. The K_m for -leucine depended on the concentration of salt and increased with decreasing salt concentration. The N. magadii enzyme was unique in its halophilicity among leucine dehydrogenases studied so far.     

Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria. II. Duroquinone reductase activity

1. Enzymatic reduction of 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) by NADH can be used in an assay procedure for the NADH dehydrogenase. The reduction of this quinone occurs in the region of the electron transport system between the primary dehydrogenase and the cytochrome system as defined by the almost complete loss of reductase activity following piericidin A treatment.

2. Duroquinone reduction can be distinguished from ubiquinone 2 reduction by the marked inhibition of the former following phospholipase C, poly- -lysine, or chloroquine diphosphate treatment. In addition, duroquinone reduction requires the presence of endogenous ubiquinone 10 specifically whereas ubiquinone 2 reduction does not require the presence of endogenous quinone. These observations are consistent with the nonequivalency of the reduction sites of duroquinone and ubiquinone 2.

3. Duroquinol can be utilized as an electron donor for the energy-linked reduction, of NAD⁺. Duroquinol reduction of NAD⁺ is dependent upon the presence of ATP, is inhibited by oligomycin, carbonyl cyanide p-trifluoro methoxyphenylhydrazone and piericidin A, and is not inhibited by antimycin A at levels which inhibit electron transport.

4. Duroquinone reduction as well as ubiquinone 2 reduction are inhibited almost completely by phospholipase A, p-chloromercuribenzoate, o-phenanthroline, and Triton X100 treatments.     

An artificial redox coenzyme based on a triazine dye template

A series of nicotinamide-containing compounds based on the structure of a triazine dye, C.I. Reactive Blue 2, which is known to interact at the coenzyme-binding sites of several NAD(P)(H)-dependent dehydrogenases,^1,2 were designed, synthesized, and characterized. The preparation of these compounds is described. Reduction of the coenzyme mimics with sodium borohydride led to an increase in absorption at 356 nm, analogous to the behavior of the natural coenzyme, NAD⁺.³ When incubated with horse liver alcohol dehydrogenase and ethanol at 25°C and pH 9.0, one of the mimics, Blue N-3, was converted into a new compound with an increased absorption at 356 nm and an R_f value on thin-layer chromatography identical to that of the reduced form produced by treatment with sodium borohydride. The oxidized and reduced forms of Blue N-3 could be separated by reverse-phase ion pair high-performance liquid chromatography. This technique could be used to measure the extent of Blue N-3 reduction: Approximately 90 turnovers were calculated for each enzyme active site over a 48-h period. Gas chromatography analysis suggested that ethanol was simultaneously converted to acetaldehyde. Blue N-3 represents the first example of a new generation of potentially inexpensive, stable, and active biomimetic redox coenzymes.     

Abnormal junctional membrane structures in cardiac myocytes expressing ectopic junctophilin type 1

A gene encoding a putative ATP-dependent DNA ligase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE1094) from A. pernix encoding a 69-kDa protein showed a 39–61% identity with other ATP-dependent DNA ligases from the archaea. Normally DNA ligase is activated by NAD⁺ or ATP. There has been no report about the other activators for DNA ligase. The recombinant ligase was a monomeric protein and catalyzed strand joining on a singly nicked DNA substrate in the presence of ADP and a divalent cation (Mg²⁺, Mn²⁺, Ca²⁺ and Co²⁺) at high temperature. The optimum temperature and pH for nick-closing activity were above 70°C and 7.5°C, respectively. The ligase remained stable for 60 min of treatment at 100°C, and the half-life was about 25 min at 110°C. This is the first report of a novel hyperthermostable DNA ligase that can utilize ADP to activate the enzyme.