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.     

Nicotinamide enhances mitochondria quality through autophagy activation in human cells

Nicotinamide (NAM) treatment causes a decrease in mitochondrial respiration and reactive oxygen species production in primary human fibroblasts and extends their replicative lifespan. In the current study, it is reported that NAM treatment induces a decrease in mitochondrial mass and an increase in membrane potential (ΔΨ m ) by accelerating autophagic degradation of mitochondria. In the NAM-treated cells, the level of LC3-II as well as the number of LC3 puncta and lysosomes co-localizing with mitochondria substantially increased. Furthermore, in the NAM-treated cells, the levels of Fis1, Drp1, and Mfn1, proteins that regulate mitochondrial fission and fusion, increased and mitochondria experienced dramatic changes in structure from filaments to dots or rings. This structural change is required for the decrease of mitochondrial mass indicating that NAM accelerates mitochondrial autophagy, at least in part, by inducing mitochondrial fragmentation. The decrease in mitochondria mass was attenuated by treatment with cyclosporine A, which prevents the loss of mitochondrial membrane potential by blocking the mitochondrial permeability transition, suggesting autophagic degradation selective for mitochondria with low ΔΨ m . All these changes were accompanied by and dependent on an increase in the levels of GAPDH, and are blocked by inhibition of the cellular conversion of NAM to NAD⁺. Taken together with our previous findings, these results suggest that up-regulation of GAPDH activity may prolong healthy lifespan of human cells through autophagy-mediated mitochondria quality maintenance.     

Calmodulin and calmodulin-mediated processes in plants

Abstract. The Ca²⁺ -binding protein calmodulin is found in all plants investigated so far. The comparison of the biochemical and functional properties reveals that it is structurally conserved and functionally preserved throughout the plant and animal kingdom. Among the plant enzymes so far known to be dependent on the Ca²⁺ -calmodulin complex are NAD kinase(s), Ca²⁺ -transport ATPase, quinate: NAD⁺ oxidoreductase, soluble and membrane bound protein kinases, and H⁺ -transport ATPase. Calmodulin may play also an important role in the regulation of other cellular reactions, such as hormone-mediated processes, secretion of enzymes, and contractile mechanisms. On the basis of the NAD kinase and its regulation by light and Ca²⁺ -calmodulin, it is suggested that changes in the cellular, free Ca²⁺ concentration following stimulation may alter the metabolism of a plant cell. According to this suggestion free Ca²⁺ may act as a second messenger in plants much as it does in animal cells.     

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⁺     

Dietary restriction involves NAD+‐dependent mechanisms and a shift toward oxidative metabolism

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD⁺‐dependent deacylases known as sirtuins, and increases in NAD⁺ that derive from higher levels of respiration. Here, we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens [DAF‐16 (FOXO), SKN‐1 (Nrf1/2/3), PHA‐4 (FOXA), AAK‐2 (AMPK)]. Complete DR lifespan extension required the sirtuin SIR‐2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC‐1, a key NAD⁺ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc‐1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD⁺‐dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD⁺ salvage. Under DR conditions, NAD⁺‐dependent processes may be supported by a DR‐induced shift toward oxidative metabolism rather than an increase in total respiration.     

Effects of cold on NAD+ kinase activity in winter rape plants

Low temperature (2°C) caused an increase in the activity of NAD⁺ kinase (EC 2.7.1.23) in leaves of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górezański). The enzyme activity markedly increased between day 4 and 11 of plant exposure to cold, then tended to decrease. Changes in activity of NAD⁺ kinase coincided with the previously observed changes in the levels of pyridine nucleotides, NADP(H) (U. Maciejewska and A. Kacperska, Physiol. Plant. 69: 687–691, 1987). As a result of cold treatment, Ca²⁺–calmodulin–dependent and Ca²⁺–calmodulin–independent NAD⁺ kinase activities increased to almost the same extent. It seems therefore, that the cold–induced activation of NAD⁺ kinase does not depend on the Ca²⁺–calmodulin complex.     

Synaptosomal Calcium Uptake Systems: Prostaglandins Are Probably Not Involved in the Regulation of Calcium Fluxes Into and Within the Nerve Endings

Abstract: ⁴⁵Ca²⁺ uptake measurements were performed on intact and osmotically lysed synaptosomes from rat brain to study the possible influence of prostaglandins (PGs) on Ca²⁺ movements into and within the nerve endings. The K⁺-induced ⁴⁵Ca²⁺ uptake of intact synaptosomes was not influenced by several inhibitors of PG synthesis. ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations was promoted by ATP and seemed to be largely attributable to mitochondria, as it was inhibited by mitochondrial poisons. This Ca²⁺ uptake was strongly reduced by PG synthesis inhibitors but also by PG precursor fatty acids. Both PG synthesis inhibitors and precursors, according to their relative efficacy in blocking Ca²⁺ uptake, were able to induce Ca²⁺ efflux from preloaded intrasynaptosomal organelles. The PGs E₂, F_2α, D₂, and thromboxane B₂ were without effect on ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations. On the basis of our results it does not seem likely that PGs influence Ca²⁺ availability by modulating Ca²⁺ fluxes into or within the nerve endings. The observed inhibitory effects of PG synthesis inhibitors and precursors on the intrasynaptosomal Ca²⁺ uptake might be due to unspecific impairment of mitochondrial functions.     

NAD+-glycohydrolase from Streptococcus pyogenes shows cyclic ADP-ribose forming activity

Abstract NAD⁺-glycohydrolase from Streptococcus pyogenes was purified by successive chromatography on CM Sepharose CL-6B, Sephacryl S-200 HR and hydroxyapatite. The purified enzyme possessed synthesis and hydrolysis activities of cyclic ADP-ribose (cADPR), a newly found second messenger for Ca²⁺ mobilisation, along with cleavage activity of the ribose-nicotinamide bond in NAD⁺.     

Quantitation of NAD+: Why do we need to measure it?

Background

Nicotinamide adenine dinucleotide (NAD⁺) is an essential pyridine nucleotide that is currently investigated as an important target to extend lifespan and health span. Age-related NAD+ depletion due to the accumulation of oxidative stress is associated with reduced energy production, impaired DNA repair and genomic instability.

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

In vitro and in vivo regulation of chloroplast glyceraldehyde-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. II. In vitro modulation of the isozyme pattern

Isozymes of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13) from Chenopodium rubrum were separated using inverse discontinuous ammonium sulphate gradient solubilization. Leaves were extracted at the 9th h of light and the 9th h of darkness of a 12 h light/12 h dark cycle. The ratio of "NADP-GPD I" to "NADP-GPD II" varied with the light/dark cycle. However, the "light" isozyme pattern could be obtained from "dark" plants by including NADP ⁺ or NAD ⁺ kinase in the extraction buffer. Similarly, the "dark" isozyme pattern was produced in "light" plants extracted in the presence of NAD⁺. Pyridine nucleotides had no effect on the separated, purified isozymes. It is concluded that differential binding of the isozymes at the moment of extraction to pelletable material in the crude extract determines the isozyme pattern, and that this binding is regulated by the pyridine nucleotide ratio.     

Calcium in fungi

Abstract. Recently much experimental evidence has accumulated concerning intracellular calcium and its fundamental role as a regulator in eukaryotic cells. The literature relating to Ca²⁺ in fungi is large and diverse and this paper draws together the available information and discusses the particular functions of the ion in this group of organisms.
Uptake mechanisms in fungi are considered with special reference to the effect of Ca²⁺ on permeability and the systems responsible for transport of ions, sugars and amino acids. Discussion of the subcellular locations and distribution of Ca²⁺ is accompanied by a critique of methodology used in determination of subcellular sites of Ca²⁺ in fungi. The role of Ca²⁺ in morphogenesis in fungi is considered with particular reference to selected groups.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

Effects of water stress on enzymes of ammonia assimilation in root nodules of alfalfa (Medicago sativa)

Protein content and activities of the enzymes glutamine synthetase (EC 6.3.1.2), NADH-glutamate synthase (EC 1.4.1.14), NADH-glutamate dehydrogenase (reductive amination (EC 1.4.1.2) and NAD⁺-glutamate dehydrogenase (oxidative deamination) (EC 1.4.1.2) from the plant fraction of root nodules of alfalfa ( Medicago sativa L. cv. Aragon) were determined under water stress. Only NADH-glutamate synthase activity was inhibited during drought. The results indicate that the glutamine synthetase/NADH-glutamate synthase cycle was fully operational in alfalfa nodules of control or even mildly stressed plants when N₂-fixation was not inhibited, but that the coupling between glutamine synthetase and NADH-glutamate synthase was lost as drought progressed. Patterns of glutamine synthetase and NADH-/NAD⁺-gluta-mate dehydrogenase activities reflect changes in ammonia content of nodules and/or availability of carbon substrates, and indicate that nodules maintain sufficient enzyme activity for ammonia assimilation throughout water stress.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

Acquired temperature-sensitive paralysis as a biomarker of declining neuronal function in aging Drosophila

General locomotor activity decreases with normal aging in animals and could be partially explained by decreases in neuronal function. Voltage-gated Na⁺ channels are essential in initiating and propagating rapid electrical impulses underlying normal locomotor activity and behavior in animals. Isolation of mutations conferring temperature-sensitive (ts) paralysis has been an extremely powerful paradigm for identifying genes involved in neuronal functions, such as membrane excitability and synaptic transmission. For instance, decreased expression of wild-type Na⁺ channels in flies harboring the no-action-potential ( nap ) mutant allele ( mle^napts ) confers rapid and reversible ts paralysis, because of failure of action potential propagation. Here, we report that aging wild-type Drosophila gradually develops an acquired susceptibility to ts paralysis that is indistinguishable from that seen in young ts paralytic mle^napts mutants. Moreover, we show that this general age-dependent susceptibility is also present in mle^napts flies, although the effects are shifted to lower temperature regimes. The mle^napts flies also exhibit decreased lifespan and increased frailty. Paralysis and decreased lifespan of mle^napts flies were partially rescued by increasing the dosage of para , the structural gene for the major action potential Na⁺ channel in central nervous system of Drosophila . Lastly, we show a dramatic scaling of ts paralysis susceptibility with chronological age in short-lived and long-lived mutant flies, further demonstrating that this age-dependent risk is independent of genetic background. Thus, decreased neural transmission, a hallmark of which is ts paralysis, is a biomarker of aging.     

Demonstration of a K+/H+ exchange activity in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Abstract Washed cells of Rhodopseudomonas sphaeroides forma sp. denitrificans , grown under photodenitrifying conditions, exhibited K⁺ uptake dependent on the transmembrane proton gradient (Δ pH). These cells also acidified the suspension medium in response to K⁺ pulses both aerobically and anaerobically in light and in the dark. The results indicate that the photodenitrifier has a reversible K⁺/H⁺ exchange activity which reflects its role in regulating the intracellular K⁺ concentration, as well as intracellular pH. The acidification of the external medium resulting from K⁺ pulses was inhibited by carbonyl cyanide- m -chlorophenylhydrazone (CCCP) indicating that the antiporter is energy-dependent. Addition of KCl to washed cells depolarized the membrane potential (Δψ) with a concomitant increase in ΔpH, indicating that the K⁺/H⁺ antiporter was electrogenic.     

酵母Sirtuins在细胞寿命中的作用

酿酒酵母(以下简略为酵母)作为寿命分析模型广泛应用于寿命研究领域。酵母寿命分析方法有两种,分别是复制型酵母寿命分析法和时序型酵母寿命分析法。目前,通过酵母寿命分析模型已识别出包括SIR2在内的多个寿命调节基因。SIR2是目前较好的被确立起来的寿命调节基因,具有NAD依赖型脱乙酰化酶的活性,从原核生物到真核生物都有良好的保守性。Sirtuins (Sir2蛋白家族的总称)在细胞内具有功能上的多样性,其中包括对于压力耐受的调节、基因转录的调节、代谢通路的调节以及寿命调节作用等。Sir2是Sirtuins家族最早发现的成员,其功能是参与异染色质结构域转录的沉默调节,同时还参与复制型酵母寿命的调节。已证明,SIR2的缺失会缩短酵母的寿命,基因表达的增高会延长寿命。Sir2的高等真核生物的同源蛋白也被证实参与衰老相关疾病的调节。本文中,我们将阐述Sir2以及Sir2的酵母同源蛋白Hst1-Hst4的功能,以及由它们调节的酵母寿命。     

Influence of cardiac-specific overexpression of insulin-like growth factor 1 on lifespan and aging-associated changes in cardiac intracellular Ca2+ homeostasis, protein damage and apoptotic protein expression

A fall in circulating levels of cardiac survival factor insulin-like growth factor 1 (IGF-1) contributes to cardiac aging. To better understand the role of IGF-1 in cardiac aging, we examined the influence of cardiac IGF-1 overexpression on lifespan, cardiomyocyte intracellular Ca²⁺ homeostasis, protein damage, apoptosis and expression of pro- and anti-apoptotic proteins in young and old mice. Mouse survival rate was constructed by the Kaplan–Meier curve. Intracellular Ca²⁺ was evaluated by fura-2 fluorescence. Protein damage was determined by protein carbonyl formation. Apoptosis was assessed by caspase-8 expression, caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay. Pro- and anti-apoptotic proteins including Bax, p53, pp53, Bcl2, Omi/HtrA2, apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) were assessed by Western blot. Aging decreased plasma in IGF-1 levels, elevated myocyte resting intracellular Ca²⁺ levels, reduced electrically stimulated rise in intracellular Ca²⁺ and delayed intracellular Ca²⁺ decay associated with enhanced protein carbonyl formation, caspase-8 expression and caspase-3 activity in FVB mice, all of which with the exception of elevated resting intracellular Ca²⁺ were attenuated by IGF-1. Aging up-regulated expression of Bax, Bcl2 and ARC, down-regulated XIAP expression and did not affect p53, pp53 and Omi/HtrA2. The IGF-1 transgene attenuated or nullified aging-induced changes in Bax, Bcl2 and XIAP. Our data suggest a beneficial role for IGF-1 in aging-induced survival, cardiac intracellular Ca²⁺ homeostasis, protein damage and apoptosis possibly related to pro- and anti-apoptotic proteins.