Littérature scientifique sur le sujet « NADH-dehydrogenase activity »

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.

ABSTRACT The reoxidation of NADH generated in reactions within the mitochondrial matrix of Saccharomyces cerevisiae is catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem. 195:857–862, 1991). Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p. Disruption of the gene designated NDH1 results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH. Disruption of a closely related gene designated NDH2 has no effect on these properties. Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol. Codisruption of NDH1 and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions.

ABSTRACTWe previously isolated respiratory-deficient mutant (RDM) strains ofZymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochromebd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within thendhgene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.

NADH dehydrogenase of Bacillus megaterium was isolated from the sonicate soluble fraction. The enzyme was purified approximately 61-fold by a combination of ammonium sulfate fractionation and column chromatography on DEAE-Sephadex and hydroxyapatite. The purified enzyme has an apparent molecular weight of 42 000 as determined by SDS-polyacrylamide gel electrophoresis and activity staining for NADH-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) oxidoreductase. The enzyme is specific for NADH and has a pH optimum of 7.5–7.8. The apparent Km values for NADH are 15.7, 34.8, and 69.2 μM as determined for NADH-DCIP (dichlorophenol–indophenol), NADH-ferricyanide, and NADH-MTT oxidoreductases. FAD is the prosthetic group of the enzyme. NAD+ acts as a competitive inhibitor. The inhibition studies suggest that NADH dehydrogenase is the primary electron donor of the NADH oxidase system. Localization studies and inhibition studies together indicate that the NADH oxidase is a complex of membrane-bound enzymes and coenzymes.Key words: NADH dehydrogenase, NADH oxidase, Bacillus megaterium, purification, characterization.

Mitochondrial NADH-dehydrogenase, succinate dehydrogenase and cytochrome oxidase activities of peripheral blood leukocytes of rats with the grafted Guerin's carcinoma were studied in the dynamics of oncogenesis under the conditions of the preliminary low-level irradiation. Tumor growth was accompanied by a decrease in NADH-dehydrogenase activity, an increase of succinate dehydrogenase activity. Cytochrome oxidase activity of leucocytes remained at the control level up to the terminal stages of tumor growth. Preliminary low-level irradiation of the tumor bearing animals caused a tendency to the decrease of enzymatic activities studied. This tendency was observed from the initial stages of oncogenesis.

ABSTRACT Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of Mycobacterium tuberculosis infections. Resistance to INH in clinical isolates has been correlated with mutations in the inhA, katG, andahpC genes. In this report, we describe a new mechanism for INH resistance in Mycobacterium smegmatis. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of theM. tuberculosis complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD+ ratio confers INH resistance.

ABSTRACT In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, l-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes thel-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.

Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide ([Formula: see text]) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving [Formula: see text]. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2(hypoxia) for 24 h followed by air-5% CO2for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of α-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess [Formula: see text] produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.

1. An NADH-dependent nitroreductase from the inner membrane of ox liver mitochondria copurified with Complex I of the respiratory redox chain (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). 2. The corresponding nitroreductase from ox heart mitochondria co-purified with the NADH-cytochrome c reductase of Mahler, Sarkar & Vernon [(1952) J. Biol. Chem. 199, 585-597] [NADH: (acceptor) oxidoreductase, EC 1.6.99.3], a component of Complex I that contains the FMN. 3. The mitochondrial nitroreductase activity is attributed to the flavoprotein component of Complex I.

L’atrophie musculaire, qui constitue un enjeu majeur en matière de santé publique, est une affection qui touche les personnes âgées, mais aussi les personnes sédentaires, immobilisées ou souffrant d’une inflammation chronique. L’utilisation de modèles animaux, en particulier les rongeurs de laboratoire, a permis d’élucider les mécanismes moléculaires et physiopathologiques à l’origine de l’atrophie musculaire. Dans la recherche de solutions thérapeutiques, l’exploration d’un modèle de résistance naturelle à l’atrophie musculaire doit permettre d’ouvrir de nouvelles pistes de recherche innovantes. Le laboratoire explore comment l‘ours brun hibernant est capable de préserver son tissu musculaire durant plusieurs mois d’immobilité, et comment son sérum est capable d’induire des modifications de la balance protéique sur des cellules musculaires humaines. L’objectif principal de mon travail de thèse était d’identifier des composés ou familles de composés circulants chez l’ours en hibernation, et responsables d’effets biologiques sur des cellules humaines. Dans un premier temps j’ai recherché une activité biologique facilement mesurable, et qui pourrait être utilisée pour cribler les composés circulants. La mesure de l’activité NADH déshydrogénase par un test colorimétrique, permet de suivre les effets inhibiteurs du sérum et de ses fractions sur des cellules humaines en culture, de façon robuste et reproductible. Grace à cet outil, nous avons pu initier le criblage de plusieurs fractions issues du sérum d’ours hibernant, débutant ainsi une approche sans a priori dans la recherche des composés actifs du sérum d’ours hibernant. Ces travaux ouvrent la voie aux tests de nouvelles fractions, permettant d’avancer vers l’identification de nouvelles molécules ayant un effet positif sur la balance énergétique cellulaire. Selon la même démarche, le développement de plusieurs outils de mesure couvrant d’autres domaines du métabolisme cellulaire devrait permettre à l’avenir de compléter cette approche. En parallèle, dans la recherche de composés circulants actifs présent dans le sérum d’ours hibernant, j’ai axé mes recherches sur des composés en relation avec le système endocannabinoïde. J’ai pu ainsi mettre en évidence une diminution globale du tonus endocannabinoïde, avec une diminution des ligands de la voie canonique. De façon surprenante, la concentration d’oleoylethanolamide (OEA) circulante est multipliée par trois en hiver, suggérant un rôle important de ce composé dans la physiologie de l’hibernation chez l’ours brun. La poursuite de ces travaux doit permettre de mieux cerner les composés circulants d’intérêt pour la médecine humaine, et d’avancer vers des solutions thérapeutiques innovantes dans la lutte de certaines pathologies, comme l’atrophie musculaire
Muscle atrophy, which is a major public health issue, is a condition that affects the elderly, but also people who are sedentary, immobilized or suffering from chronic inflammation. The use of animal models, in particular laboratory rodents, has made it possible to elucidate the molecular and physiopathological mechanisms at the origin of muscle atrophy. In the search for therapeutic solutions, the exploration of a model of natural resistance to muscle atrophy should open up new and innovative avenues of research. The laboratory is exploring how the hibernating brown bear is able to preserve its muscle tissue during several months of immobility, and how its serum is able to induce changes in the protein balance of human muscle cells. The main objective of my thesis work was to identify compounds or families of compounds circulating in the hibernating bear and responsible for biological effects on human cells. First, I looked for a biological activity that could be easily measured and that could be used to screen the circulating compounds. The measurement of NADH dehydrogenase activity by a colorimetric assay, allows to follow the inhibitory effects of serum and its fractions on human cells in culture, in a robust and reproducible way. Thanks to this tool, we were able to initiate the screening of several fractions from hibernating bear serum, thus starting an unbiased approach in the search for active compounds in hibernating bear serum. This work opens the way to the testing of new fractions, allowing to advance towards the identification of new molecules having a positive effect on the cellular energy balance. According to the same approach, the development of several measurement tools covering other domains of cellular metabolism should allow to complete this approach in the future. In parallel, in the search for active circulating compounds present in the serum of hibernating bears, I focused my research on compounds related to the endocannabinoid system. I was thus able to highlight a global decrease of the endocannabinoid tone, with a decrease of the ligands of the canonical pathway. Surprisingly, the concentration of circulating oleoylethanolamide (OEA) is multiplied by three in winter, suggesting an important role of this compound in the physiology of hibernation in brown bears. The continuation of this work should allow to better identify circulating compounds of interest for human medicine, and to advance towards innovative therapeutic solutions in the fight against certain pathologies, such as muscle atrophy

Recemment, deux genes (nuo8, nuo9) de la bacterie photosynthetique rhodobacter capsulatus, codant des proteines analogues aux sous-unites nd1 et tyky de la nadh:ubiquinone oxydoreductase bovine (complexe l) ont ete clones. Une mini-banque d'adn genomique de la bacterie r. Capsulatus a ete construite dans le vecteur lamdagem-11 pour explorer les regions de l'adn adjacentes aux genes nuo8 et nuo9. Le dechiffrage de la sequence d'un fragment d'adn de 4. 915 pb, localise a portee immediate du gene nuo9, a permis de mettre en evidence 4 genes (nuo10, nuo11, nuo12, nuo13) codant des proteines hydrophobes respectivement analogues aux sous-unites nd6, nd4l, nd5 et nd4 du complexe l mitochondrial. Des souches mutantes de r. Capsulatus ont ete obtenues par insertion d'une cassette de resistance a la kanamycine dans les genes nuo8 et nuo12. Ces mutations retentissent sur le developpement de la bacterie: le developpement aerobie est fortement ralenti, le developpement photoheterotrophique necessite du co#2, les bacteries ne se developpent pas en photoautotrophie. La caracterisation polarographique et biochimique des souches mutantes montre l'absence totale d'activite nadh:ubiquinone oxydoreductase. Les spectres rpe, realises sur les membranes des souches mutantes reduites par le dithionite montrent l'absence des signatures caracteristiques des centres fe-s du complexe l de la bacterie. Ces resultats demontrent que le locus nuo de r. Capsulatus, code pour la nadh:ubiquinone oxydoreductase de la bacterie. La destruction de l'un ou l'autre des genes nuo8 et nuo12 entraine l'absence d'assemblage du complexe l dans la membrane bacterienne. Le retentissement des mutations sur le developpement bacterien est attribue a des perturbations de la balance redox

Abstract Pyruvate dehydrogenase complexes (PDCs) are large multi-enzyme complexes which catalyse the oxidative decarboxylation of pyruvate to yield acetyl-CoA, CO, and NADH. Component enzymes of all PDCs are pyruvate dehydrogenase (PDH, El), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Plants are unique in having discrete PDCs in two organelles, mitochondria and plastids (Randall et al. 1989). Most mitochondrial complexes are regulated by reversible phosphorylation and also have two associated regulatory enzymes, PDH kinase and PDH-P phosphatase (Reed 1981). The regulatory enzymes are not associated with yeast mitochondrial PDCs (mtPDCs) or with the higher plant plastid form of PDC. The primary function of plant mtPDC is to provide acetylCoA for the Krebs cycle and NADH for oxidative phosphorylation, while the plastid PDC is a primary source of acetyl-CoA and NADH for fatty acid and isoprenoid biosynthesis. Our investigations of the mtPDC in plants are centred on establishing the regulatory mechanisms that control its activity, particularly during photosynthesis and photorespiration. These efforts have provided the first proof that activity of a plant enzyme could be regulated by reversible phosphorylation (Randall and Rubin 1977; Randall et al. 1981; Rao and Randall 1980). Phosphorylation inactivates mtPDC, which can then be reactivated by dephosphorylation (Randall et al. 1989). Thus, reversible phosphorylation is an off-on switch for the plant mtPDC as it is in most higher eukaryotes.

Numerous enzymes, including those of the hexose monophosphate and glycolytic pathways, are active in the red cell. They are required for the generation of ATP and the reductants NADH and NADPH. 2,3-Diphosphoglycerate, an intermediate of glucose metabolism, is a key regulator of the affinity of haemoglobin for oxygen, and accessory enzymes are also active for the synthesis of glutathione, disposal of oxygen free radicals, and for nucleotide metabolism. With the exception of heavy metal poisoning and rare cases of myelodysplasia, most red cell enzyme deficiency disorders are inherited. They may cause haematological abnormalities, (most commonly nonspherocytic haemolytic anaemias, but also rarely polycythaemia or methaemoglobinaemia, manifest with autosomal recessive or sex-linked inheritance), and may also be associated with nonhaematological disease when the defective enzyme is expressed throughout the body. Some may mirror important metabolic disorders, without producing haematological problems, making them of diagnostic value. Others are of no known clinical consequence. With rare exceptions, it is impossible to differentiate the enzymatic defects from one another by clinical or routine laboratory methods. Diagnosis depends on the combination of (1) accurate ascertainment of the family history; (2) morphological observations—these can determine whether haemolysis is present, rule out some causes of haemolysis (e.g. hereditary spherocytosis and other red blood cell membrane disorders), and diagnose pyrimidine 5′-nucleotidase deficiency (prominent red cell stippling); (3) estimation of red cell enzyme activity; and (4) molecular analysis. The most common red cell enzyme defects are glucose-6-phosphate dehydrogenase deficiency, pyruvate kinase deficiency, glucose-6-phosphate isomerase deficiency, pyrimidine 5′-nucleotidase deficiency—which may also induced by exposure to environmental lead—and triosephosphate isomerase deficiency.