Genetically encoded fluorescent sensors for intracellular NADH detection

We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.     

Frex and FrexH: Indicators of metabolic states in living cells

Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form play central roles in energy and redox metabolisms. For many years, researchers have relied on the weak NADH endogenous fluorescence signal to determine the NADH level in living cells. We recently reported a series of genetically encoded fluorescent sensors highly specific for NADH. These sensors allow real-time, quantitative measurement of this significant molecule in different subcellular compartments. In this study, we provide a more detailed discussion of the benefits and limitations of these genetically encoded fluorescent sensors. These sensors are utilized in most laboratories without the need for sophisticated instruments because of their superior sensitivity and specificity. They are also viable alternatives to existing techniques for measuring the endogenous fluorescence of intracellular NAD(P)H.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

New approaches to NAD(P)H regeneration in the biosynthesis systems

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), as two kinds of well-known cofactor, are widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. In general, supply of NAD(P)H is a major challenged factor in redox fermentation systems due to its high cost and low stability, which have stimulated the development of NADH regeneration systems in recent years. Until now, a series of NAD(P)H regeneration systems have been developed. This review focuses primarily on new approaches of NAD(P)H cofactor regeneration in the biosynthesis systems, such as single cell in vivo NADH regeneration system, double cell coupling NADH regeneration system, in vitro enzyme-coupled NADH regeneration system, microbial cell surface display NADH regeneration system. Finally, the prospect and tendency of NADH regeneration are discussed.     

Disorders in NADPH generation via pentose phosphate pathway influence the reproductive potential of the Saccharomyces cerevisiae yeast due to changes in redox status

Intermediary metabolites have a crucial impact on basic cell functions. There is a relationship between cellular metabolism and redox balance. To maintain redox homoeostasis, the cooperation of both glutathione and nicotine adenine dinucleotides is necessary. Availability of nicotinamide adenine dinucleotide phosphate (NADPH) as a major electron donor is critical for many intracellular redox reactions. The activity of glucose-6-phosphate dehydrogenase (Zwf1p) and 6-phosphogluconate dehydrogenase (Gnd1p and Gnd2p) is responsible for NADPH formation in a pentose phosphate (PP) pathway. In this study, we examine the impact of redox homoeostasis on cellular physiology and proliferation. We have noted that the Δzwf1 mutant lacking the rate-limiting enzyme of the PP pathway shows changes in the cellular redox status caused by disorders in NADPH generation. This leads to a decrease in reproductive potential but without affecting the total lifespan of the cell. The results presented in this paper show that nicotine adenine dinucleotides play a central role in cellular physiology.     

(Semi-)quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart.

In vivo analysis of the metabolic state of tissue by means of reduced nicotinamide adenine dinucleotide (NADH) fluorimetry is disturbed by tissue movements and by hemodynamic and oximetric effects. These factors cause changes in the absorption of ultraviolet (UV) excitation light by the tissue. Many different methods have been used in the literature to compensate measured NADH fluorescence intensities for these effects. In this paper we show on theoretical grounds that the ratio of NADH fluorescence intensity and UV diffuse reflectance intensity provides a (semi-)quantitative measure of tissue NADH concentrations. This result is corroborated by experiments with tissue phantoms in which absorption and back-scattering properties were varied. Furthermore, we have verified the validity of this compensation method in isolated Langendorff-perfused rat heart preparations. In this preparation oximetric effects (of blood and tissue) are the major determinants of the metabolism-dependent UV diffuse reflectance change. Hemodynamic effects accompanying compensatory vasodilation are negligible. Movement artifacts were eliminated by simultaneously recording fluorescence and reflectance images, using a CCD camera with a biprism configuration. The results show that the NADH fluorescence/UV reflectance ratio can be used to monitor the mitochondrial redox state of the surface of intact blood-perfused myocardium.     

High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as approximately 10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 microg/ml alamethicin and the reaction is terminated by H(2)SO(4), which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1-20 microM. NADH is quantitatively analyzed with a detection limit of 3-5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.     

Determination of the metabolic index using the fluorescence lifetime of free and bound nicotinamide adenine dinucleotide using the phasor approach

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However, in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is a subject of debate. The fluorescence lifetime of NADH increases when bound to an enzyme, in contrast to the short multiexponential lifetime displayed by NADH in solution. The extent of fluorescence lifetime increase depends on the enzyme to which NADH is bound. With proper preparation of lactate dehydrogenase (LDH) using oxalic acid (OA) as an allosteric factor, bound NADH to LDH has a lifetime of 3.4 ns and is positioned on the universal semicircle of the phasor plot, inferring a monoexponential lifetime for this species. Surprisingly, measurements in the cellular environments with different metabolic states show a linear trajectory between free NADH at about 0.37 ns and bound NADH at 3.4 ns. These observations support that in a cellular environment, a 3.4 ns value could be used for bound NADH lifetime. The phasor analysis of many cell types shows a linear combination of fractional contributions of free and bound species NADH.     

Determination of NADH in frozen rat brain sections by laser-induced fluorescence

Methods to assess metabolism are important analytical tools in neuroscience. The fluorophore nicotinamide adenine dinucleotide (NADH) is a parameter of cellular metabolism. NADH fluorescence was measured using a laser-based fluorescence detector with spectral and temporal filters. Distribution and intensity of NADH fluorescence were investigated in frozen brain sections. In sections containing hippocampus the intensity of NADH fluorescence was correlated to brain structures. In order to investigate the consequences of neurotoxic lesions, 5,7-dihydroxytryptamine was injected into the dorsal raphe nucleus 4 to 240 days prior to the measurement. NADH fluorescence decreased in the affected region by 50%, indicating that no recovery in metabolic activity had occurred.     

Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells

Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time‐resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC‐5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue‐ and red‐shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra‐cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.     

Coupling of redox indicator dyes into an enzymatic reaction cycle

The spectral properties of ten redox indicator dyes were evaluated with the aim of finding the optimal choice for coupling to enzymatic reactions with high sensitivity for the production of the reduced form. Eight of the dyes were selected for coupling into a reaction cycle formed by yeast alcohol dehydrogenase with substrates ethanol and nicotinamide adenine dinucleotide (NAD+) and diaphorase with substrates reduced nicotinamide adenine dinucleotide (NADH, produced by the prior reaction) and the oxidized form of the respective dye. Two of the dyes exhibited decreased absorption on reduction, whereas all (eight) tetrazolium dyes increased in their absorption substantially upon reduction. Bis-tetrazolium dyes had a significantly higher molar extinction coefficient (up to 23,000 M-1.cm-1) than mono-tetrazolium dyes (down to 8000 M-1.cm-1). Kinetically, most dyes could be reduced with NADH (and diaphorase), but the rate of reduction varied considerably among the dyes with nitroblue tetrazolium (NBT) and tetranitroblue tetrazolium (TNBT) being the fastest. Therefore, NBT and TNBT seem to be the most suitable for fast response.     

An alcohol dehydrogenase ribozyme

We report an RNA molecule that exhibits activity analogous to that of alcohol dehydrogenase (ADH). Directed in vitro evolution was used to enrich nicotinamide adenine dinucleotide (NAD+)-dependent redox-active RNAs from a combinatorial pool. The most active ribozyme in the population forms a compact pseudoknotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. Moreover, this ADH RNA was coupled with a redox relay between NADH and flavin adenine dinucleotide to give a NAD+-regeneration system. Our demonstration of the redox ability of RNA adds support to an RNA-based metabolic system in ancient life.     

Purification and Characterization of the Two 6-Phosphogluconate Dehydrogenase Species from Pseudomonas multivorans

The two species of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) from Pseudomonas multivorans were resolved from extracts of gluconate-grown bacteria and purified to homogeneity. Each enzyme comprised between 0.1 and 0.2% of the total cellular protein. Separation of the two enzymes, one which is specific for nicotinamide adenine dinucleotide phosphate and the other which is active with nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was facilitated by the marked difference in their respective isoelectric points, which were at pH 5.0 and 6.9. Comparison of the subunit compositions of the two enzymes indicated that they do not share common peptide chains. The enzyme active with nicotinamide adenine dinucleotide was composed of two subunits of about 40,000 molecular weight, and the nicotinamide adenine dinucleotide phosphate-specific enzyme was composed of two subunits of about 60,000 molecular weight. Immunological studies indicated that the two enzymes do not share common antigenic determinants. Reduced nicotinamide adenine dinucleotide phosphate strongly inhibited the 6-phosphogluconate dehydrogenase active with nicotinamide adenine dinucleotide by decreasing its affinity for 6-phosphogluconate. Guanosine-5'-triphosphate had a similar influence on the nicotinamide adenine dinucleotide phosphate-specific 6-phosphogluconate dehydrogenase. These results in conjunction with other data indicating that reduced nicotinamide adenine dinucleotide phosphate stimulates the conversion of 6-phosphogluconate to pyruvate by crude bacterial extracts suggest that in P. multivorans, the relative distribution of 6-phosphogluconate into the pentose phosphate and Entner-Doudoroff pathways might be determined by the intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate and purine nucleotides.     

A fundamental regulatory role of formate on thuringiensin production by resting cell of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> YBT-032

In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed. The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability.     

Single-sample preparation for simultaneous cellular redox and energy state determination

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-microm-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma.     

Biochemical Basis of Obligate Autotrophy in Nitrosomonas europaea

The specific activities of isocitric dehydrogenase, alpha-ketoglutaric dehydrogenase, succinic dehydrogenase, malic dehydrogenase, and reduced nicotinamide adenine dinucleotide (NADH) oxidase were determined in extracts of Nitrosomonas europaea and compared with the corresponding values for Anacystis nidulans and autotrophically grown Hydrogenomonas eutropha. In common with other obligate autotrophs and in contrast to facultative autotrophs, Nitrosomonas extracts lacked alpha-ketoglutaric dehydrogenase and KCN-sensitive NADH oxidase activity and had low succinic dehydrogenase activity. The Nitrosomonas NADH oxidase appeared to be of the peroxidase type.     

Preparation and characterization of the NAD vinylogue, 3-pyridylacryloamide adenine dinucleotide

The vinylogue of NAD, 3-pyridylacryloamide adenine dinucleotide, was prepared from NAD and 3-pyridylacryloamide through the snake venom NADase-catalyzed transglycosidation reaction. The analog, purified by ion-exchange chromatography, was obtained in a 55% yield. The cyanide adduct and reduced form of the analog exhibited absorbance maxima at 358 nm and 378 nm, respectively, with extinction coefficients in each case being 2.3-times higher than those reported for the corresponding NAD derivatives. 3-Pyridylacryloamide adenine dinucleotide served as a coenzyme with bovine liver glutamic dehydrogenase and to a lesser extent with malate and lactate dehydrogenases. The analog was not reduced in reactions catalyzed by yeast and horse liver alcohol dehydrogenases, sheep liver sorbitol dehydrogenase, and rabbit muscle glycerophosphate dehydrogenase. Substitution of the pyridylacryloamide analogs for NAD and NADH in the assay of substrates for glutamic dehydrogenase was demonstrated.     

Integrated detection of intrinsic fluorophores in live microbial cells using an array of thin film amorphous silicon photodetectors

Two-dimensional fluorescence spectroscopy (2D FS) provides a non-invasive means to assess cell condition without the introduction of changes to the cell environment. The method relies on the measurement of the excitation-emission fluorescence intensity matrix of key intrinsic fluorophores, like aromatic amino acids, enzyme cofactors, and vitamins. Commonly used detection systems are complex, with multiple bandpass filters, and are hard to miniaturize. Here, an amorphous silicon photodetector array system integrated with amorphous silicon-carbon alloy filters designed to detect three key fluorophores - tryptophan (Trp), reduced nicotine adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) - is demonstrated. These intrinsic fluorophores were detected in pure solutions and also in suspended yeast cells. The array system was used to monitor changes in intrinsic fluorophore concentration when a yeast cell solution was subject to a thermal shock stress.     

Determination of oxidized and reduced nicotinamide adenine dinucleotide in cell monolayers using a single extraction procedure and a spectrophotometric assay

While many investigations measuring oxidized nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADH) have been carried out on several mammalian tissues and blood cells, few reports have dealt with monolayers of cultured cells. Here we show a novel method to measure NAD+ and NADH in monolayers of a neuroblastoma cell line. The method was established by modifying a single extraction procedure originally developed for erythrocytes and an enzymatic cycling assay using a dye that absorbs in visible range. The following modifications were made. (i) Addition of 0.05% of a detergent, Triton X-100, to carbonate-bicarbonate extraction buffer enabled us to accurately measure cellular [NADH]/([NAD+]+[NADH]). (ii) Addition of N-ethyldibenzopyrazine ethyl sulfate salt (phenazine ethosulfate) immediately before the incubation suppressed the gradual decline of the sensitivity of the assay. The procedure presented here provides a simple and inexpensive measurement of NAD+ and NADH in cell monolayers.     

Determination of lactate dehydrogenase isoenzymes in single rat glioma cells by capillary electrophoresis with electrochemical detection

A method for determination of lactate dehydrogenase (LDH) isoenzymes in single rat glioma cells (C6) was developed. In this method, a whole cell was electrokinetically injected into the front end of the separation capillary. After that, the cell was lysed by ultrasonication and the isoenzymes in the cell were pre-separated at 20 kV for 5 min and then incubated for 2 min with the enzyme substrates nicotinamide adenine dinucleotide (NAD(+)) and lactate in the capillary electrophoresis running buffer. The electroactive product NADH generated by the isoenzymes through on-capillary enzyme-catalyzed reaction was detected at the outlet of capillary by using the end-capillary amperometric detection with a constant potential mode at a carbon fiber bundle microdisk electrode. Since the amplification of signal via the enzyme reaction, the concentration of nicotinamide adenine dinucleotide (NADH) is much higher than that of LDH. The external standardization was used to quantify isoenzymes in individual cells. Three LDH isoenzymes in single rat glioma cells (C6) were determined and quantified.