NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP⁺, NAD⁺ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30° C. Lower but significant protection and reactivation was also observed with NADP⁺ and NAD⁺. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O₂ or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD⁺ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.Abbreviations DTNB 5,5-dithiobis(2-nitrobenzoate) - EGTA Ethylenglycoltetraacetic Acid - TNB 5-thio-2-nitrobenzoate - Trx Thioredoxin - Trx(SH)2 Reduced Thioredoxin - Trx-S2 Oxidized Thioredoxin     

Pyridine nucleotide transhydrogenases of parasitic helminths.

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD⁺ and NADH:NADP⁺ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD⁺ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD⁺ system was approximately five times more active than the NADH:NADP⁺ system. The NADH:NADP⁺ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD⁺ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD⁺ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD⁺ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD⁺ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD⁺ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD⁺ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.     

A Hydrazine Coupled Cycling Assay Validates the Decrease in Redox Ratio under Starvation in Drosophila

A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD⁺/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP⁺ and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD⁺ and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation.     

Endothelium and smooth muscle of pig coronary artery: Differences in metabolism

Here, we report that the smooth muscle and endothelium of the pig coronary artery differ in the profiles of energy metabolism nucleotides. ATP levels in the freshly isolated smooth muscle (1490 ± 93, all the values are in pmol/mg protein) were significantly greater than in the endothelium (418 ± 68). In contrast, endothelium contained higher levels of NADH (328 ± 21), NAD⁺ (1210 ± 28), NADPH (87 ± 2), and NADP⁺ (77 ± 4) than smooth muscle (17 ± 2, 96 ±14, 7 ± 1, and 8 ± 1, respectively). However, smooth muscle and endothelium do not differ from each other in the ratios of NADH/NAD⁺ or NADPH/NADP⁺. Cells cultured from smooth muscle and endothelium contained less ATP (93 ± 2, 141 ± 6) and had lower ratios of NADH/NAD⁺ than the freshly isolated tissues but the NADPH/NADP⁺ ratios remained similar. We conclude that (a) freshly isolated smooth muscle and endothelium differ in their profiles of the energy metabolism nucleotides, and (b) culturing the cells alters the profile.     

Design of a cytochrome P450BM3 reaction system linked by two‐step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenase

A cytochrome P450BM3‐catalyzed reaction system linked by a two‐step cofactor regeneration was investigated in a cell‐free system. The two‐step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD⁺‐dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD⁺) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP⁺ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3‐catalyzed reaction linked by the two‐step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10‐fold under initial reaction conditions. In contrast, a 10‐fold increase in STH units resulted in about a 9‐fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate‐determining step. In the system lacking the two‐step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p‐nitrophenoxydecanoic acid) was attained using 50 μM NADPH. In contrast, with the two‐step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD⁺ and NADP⁺) within 1 h. Furthermore, a 10‐fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD⁺ and NADP⁺. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Ferredoxin/thioredoxin system plays an important role in the chloroplastic NADP status of Arabidopsis

NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP⁺) and pool size (sum of NADPH and NADP⁺), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP⁺ ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found in vitro to inefficiently reduce m‐type thioredoxin, which activates Calvin cycle enzymes, and NADP‐dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP‐malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m‐type thioredoxin exhibited similar light‐dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m‐type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP⁺ ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size.     

Diurnal changes in adenylates and nicotinamide nucleotides in sugar beet leaves

Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25°C, with a photon flux density of 500 mol m^-2s^-1. Measurements were made of net CO₂ exchange, leaf adenylates (ATP, ADP and AMP), and leaf nicotinamide nucleotides (NAD⁺, NADP⁺, NADH, NADPH), over the diurnal period (16h light/8 h dark) and during photosynthetic induction. All the measurements were carried out on recently expanded leaves from 5-week-old plants. When the lights were switched on in the growth chamber, the rate of photosynthetic CO₂ uptake, and the levels of leaf ATP and NADPH increased to a maximum in 30 min and remained there throughout the light period. The increase in ATP over the first few minutes of illumination was associated with the phosphorylation of ADP to ATP and the increase in NADPH with the reduction of NADP⁺; subsequently, the increase in ATP was associated with an increase in total adenylates while the increase in NADPH was associated with an accumulation of NADP⁺ and NADPH due to the light-driven phosphorylation of NAD⁺ to NADP⁺. On return to darkness, ATP and NADPH values decreased much more slowly, requiring 2 to 4 hours to reach minimum values. From these results we suggest that (i) the total adenylate and NADPH and NADP⁺ (but not NAD⁺ and NADH) pools increase following exposure to light; (ii) the increase in pool size is not accompanied by any large change in the energy or redox states of the system; and (iii) the measured ratios of ATP/ADP and NADPH/NADP⁺ for intact leaves are low and constant during steady-state illumination.Abbreviations AEC adenylate energy charge - DHAP dihydroxyacetone phosphate - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide - PES phenazine ethosulfate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - PFD photon flux density - Ru5P ribulose-5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

Interactions of calcium with the pyridine nucleotides

Association constants were determined for the 1:1 interactions of calcium with NAD⁺, NADH, NADP⁺, and NADPH in aqueous systems (pH 7, 25 °C) by use of a calcium-sensitive electrode. The order of binding of calcium to these pyridine nucleotides appears to be NAD⁺ < NADH < NADP⁺ < NADPH with association constants of 0.2 × 10², 0.3 × 10², 0.9 × 10², and 2 × 10², respectively. Calorimetric experiments revealed that all of these interactions are endothermic with enthalpy changes of 1, 2, 2, and 3 kcal/mol, respectively.     

Inactivation of nitrate reductase by NADH in Nitrobacter agilis

Nitrate reductase from Nitrobacter agilis was inactivated by NADH (but not by NADPH) in the absence of nitrate.The inactivation of the enzyme by over-reduction with NADH was overcome by oxidizing the reduced enzyme with nitrate, ferricyanide, NAD⁺ or NADP⁺.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

Oxidation of excited-state NADH and NAD dimer in aqueous medium involvement of O2− as a mediator in the presence of oxygen

It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD⁺ (or NADP⁺). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H₂O₂ at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD⁺ is about 75%. Under analogous irradiation conditions, the dimers (NAD)₂ and (NADP)₂ undergo disproportionation to NAD⁺ and NADP⁺, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)₂.     

Inhibition of ruthenium red-insensitive mitochondrial Ca2+ release and its pyridine nucleotide specificity

Isolated, intact rat liver mitochondria, without extraneous substrates but loaded with Ca²⁺ (20 nmol/mg), can be observed to release Ca²⁺ when treated with ruthenium red. Such release can be inhibited by 0.33 mM dlisocitrate but not by 10 mM dl-β-hydroxybutyrate. Assays of NADP⁺, NADPH, NAD⁺, and NADH revealed that only the reduction of NADP⁺ can be linked with such inhibition of Ca²⁺ release, not that of NAD⁺. Since ruthenium redinsensitive Ca²⁺ release is a physiological (but normally masked) process, this experimental approach avoids some potential problems ascribed to strong pyridine nucleotide oxidation. It is suggested that specific NADP⁺:NADPH dependent reactions are part of a physiological mechanism regulating Ca²⁺ release/retention.     

Comparison of tissue preparation methods for assay of nicotinamide coenzymes

To prepare tissues for analysis of NAD⁺, NADH, NADP⁺, and NADPH, common practice is to freeze samples in liquid nitrogen, often followed by freeze-drying, before extraction in HCl or NaOH. With cucumber (Cucumis sativus L.) cotyledons, prefreezing in liquid nitrogen or slower freezing to −20°C yielded substantially lower values for NADH and NADPH than obtained from samples homogenized immediately in acid or base. Freeze-drying after freezing in liquid nitrogen generally caused even lower values of those coenzymes. We suggest that direct extraction is more likely to yield accurate results with cotyledons and other plant parts.     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K_d values) for NADPH (0.87 μM), NADP⁺ (16 μM), NADH (50 μM), and NAD⁺ (100-500 μM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K_d values for NAD⁺ and NADH are similar to those previously reported with isolated dI, but the K_d values for NADP⁺ and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidised.     

NAMPT-Mediated Salvage Synthesis of NAD+ Controls Morphofunctional Changes of Macrophages

Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD⁺ and NADH) and NADP(H) (i.e. NADP⁺ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD⁺-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD⁺ in macrophages. Inhibition of NAD⁺ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD⁺ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD⁺ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD⁺ levels by NAD⁺, NMN, or NADP⁺ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD⁺’s cytosolic role in the regulation of morphofunctional characteristics of macrophages.     

Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity

NAD⁺ and NADP⁺, chemically similar and with almost identical standard oxidation–reduction potentials, nevertheless have distinct roles, NAD⁺ serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD⁺-dependent for glutamate oxidation, NADP⁺-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD⁺ reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD⁺ but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP⁺ reduction by NADH, maintaining the coenzyme pools at different oxidation–reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD⁺-dependent, NADP⁺-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD⁺ or for NADP⁺ has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2′- and 3′-hydroxyls, dictating NAD⁺ specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD⁺ only, NADP⁺ only, or in higher animals both.     

Metabolic Regulation in Diseased Leaves II. Changes in Nicotinamide Nucleotide Coenzymes in Barley Leaves Infected With Powdery Mildew

Nicotinamide nucleotide coenzymes were estimated spectrophotometrically in noninfected barley leaves and leaves infected with Erysiphe graminis var hordei (powdery mildew). Amounts of NADH, NADP⁺ and NADPH were not altered by infection. In contrast, the NAD⁺ content rose sharply and at 144 hours was 100% greater than in noninfected leaves. The respiratory rate was increased in infected leaves and the pattern of this increase was similar to that of NAD⁺.

The effect of infection on the intracellular distribution of NADP⁺ was examined by fractionating lyophilized leaves in a nonaqueous medium. In noninfected leaves almost all of the NADP⁺ was localized in the chloroplasts. In infected leaves where some chloroplast breakdown occurs, about 60% of the NADP⁺ was detected in the nonchloroplast part of the cell. This intracellular redistribution of NADP⁺ is discussed in relation to the increased pentose-P pathway activity occuring after infection.

     

l-proline dehydrogenase of Triticum vulgare germ: Purification,properties and cofactor interactions

The enzyme catalysing the l-proline-dependent reduction of NAD⁺has been purified over 600-fold from wheat germ acetone powder extracts. l-Proline, 3,4 dehydro-dl-proline, thiazolidine-4-carboxylate were the only substrates utilized readily. The K_m for l-proline was 1·0 mM and for NAD⁺ 0·8 mM. The enzyme was highly specific for NAD⁺ with NADP⁺ and NADPH acting as effective competitive inhibitors with a K_i of 1·8 and 0·4 μM, respectively. All ribonucleoside triphosphates tested were good non-competitive inhibitors, in particular UTP. The purified enzyme could reduce pyrroline-5-carboxylate, either chemically synthesized or generated in a linked assay system from ornithine by a highly-purified ornithine transaminase. In the latter case both NADH and NADPH were utilized equally well as the reductant. With chemically synthesized dl-pyrroline-5-carboxy-late as the substrate. NADPH was used at only 25% the rate of NADH, and NADPH strongly inhibited the oxidation of NADH.     

Quantitative extraction and estimation of intracellular nicotinamide nucleotides of Escherichia coli.

The conditions of extraction of nicotinamide nucleotides from Escherichia coli were systematically investigated. The optimum pH for NAD⁺ and NADP⁺ appeared to be around 1.5 while that for NADH and NADPH was around 12.5. Seven minutes at 60°C was chosen for the extraction of all four nucleotides. Addition of CuCl to the alkaline extract improved considerably the recovery of the reduced forms. Two sensitive (10–100 pmol) assay methods for nicotinamide nucleotides were compared. A spectrophotometric cycling method based on the measurement of reduced thiazolyl blue formation appeared to be more sensitive and more reproducible and gave better recovery than a fluorometric cycling method used earlier in bacterial studies.