Journal articles on the topic 'NADH-dehydrogenase activity'
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Murray, G. I., M. D. Burke, and S. W. Ewen. "Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue." Journal of Histochemistry & Cytochemistry 36, no. 7 (July 1988): 815–19. http://dx.doi.org/10.1177/36.7.3385192.
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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.
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Small, W. Curtis, and Lee McAlister-Henn. "Identification of a Cytosolically Directed NADH Dehydrogenase in Mitochondria of Saccharomyces cerevisiae." Journal of Bacteriology 180, no. 16 (August 15, 1998): 4051–55. http://dx.doi.org/10.1128/jb.180.16.4051-4055.1998.
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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.
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Hayashi, Takeshi, Tsuyoshi Kato, and Kensuke Furukawa. "Respiratory Chain Analysis of Zymomonas mobilis Mutants Producing High Levels of Ethanol." Applied and Environmental Microbiology 78, no. 16 (June 1, 2012): 5622–29. http://dx.doi.org/10.1128/aem.00733-12.
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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.
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Thiagalingam, Sam, and Tsanyen Yang. "Purification and characterization of NADH dehydrogenase from Bacillus megaterium." Canadian Journal of Microbiology 39, no. 9 (September 1, 1993): 826–33. http://dx.doi.org/10.1139/m93-123.
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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.
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Marchenko, M. M., and O. N. Voloshchuk. "The state of the mitochondrial energy-supplying system of blood leukocytes in the dynamics of guerin's carcinoma growth under the low-level irradiation conditions." Biomeditsinskaya Khimiya 60, no. 6 (2014): 631–35. http://dx.doi.org/10.18097/pbmc20146006631.
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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.
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Huston, Scott, John Collins, Fangfang Sun, Ting Zhang, Timothy D. Vaden, Y. ‐H Percival Zhang, and Jinglin Fu. "An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation." Biotechnology and Applied Biochemistry 65, no. 3 (October 2, 2017): 286–93. http://dx.doi.org/10.1002/bab.1607.
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Miesel, Lynn, Torin R. Weisbrod, Jovita A. Marcinkeviciene, Robert Bittman, and William R. Jacobs. "NADH Dehydrogenase Defects Confer Isoniazid Resistance and Conditional Lethality in Mycobacterium smegmatis." Journal of Bacteriology 180, no. 9 (May 1, 1998): 2459–67. http://dx.doi.org/10.1128/jb.180.9.2459-2467.1998.
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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.
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Chapuy-Regaud, Sabine, Frédérique Duthoit, Laurence Malfroy-Mastrorillo, Pierre Gourdon, Nic D. Lindley, and Marie-Claude Trombe. "Competence Regulation by Oxygen Availability and by Nox Is Not Related to Specific Adjustment of Central Metabolism inStreptococcus pneumoniae." Journal of Bacteriology 183, no. 9 (May 1, 2001): 2957–62. http://dx.doi.org/10.1128/jb.183.9.2957-2962.2001.
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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.
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Powell, Charles S., and Robert M. Jackson. "Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD." American Journal of Physiology-Lung Cellular and Molecular Physiology 285, no. 1 (July 2003): L189—L198. http://dx.doi.org/10.1152/ajplung.00253.2002.
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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.
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Smyth, G. E., and B. A. Orsi. "Nitroreductase activity of NADH dehydrogenase of the respiratory redox chain." Biochemical Journal 257, no. 3 (February 1, 1989): 859–63. http://dx.doi.org/10.1042/bj2570859.
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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.
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Brass, Eric P., William R. Hiatt, Andrew W. Gardner, and Charles L. Hoppel. "Decreased NADH dehydrogenase and ubiquinol-cytochromec oxidoreductase in peripheral arterial disease." American Journal of Physiology-Heart and Circulatory Physiology 280, no. 2 (February 1, 2001): H603—H609. http://dx.doi.org/10.1152/ajpheart.2001.280.2.h603.
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Peripheral arterial disease (PAD) is associated with muscle metabolic changes that may contribute to the disability in these patients. However, the biochemical defects in PAD have not been identified. The present study was undertaken to test the hypothesis that PAD is associated with specific defects in skeletal muscle electron transport chain activity. Seventeen patients with PAD and nine age-matched controls underwent gastrocnemius muscle biopsies. There were no differences in the mitochondrial content per gram of skeletal muscle as assessed by citrate synthase activity between the PAD patients and the control subjects. Skeletal muscle NADH dehydrogenase activity was decreased by 27% compared with controls when expressed per unit of citrate synthase activity. Expression of enzyme activities normalized to cytochrome c-oxygen oxidoreductase activity confirmed a 26% decrease in NADH dehydrogenase activity and also demonstrated a 38% decrease in ubiquinol-cytochrome c oxidoreductase activity. Thus PAD is associated with specific changes in muscle mitochondrial electron transport chain activities characterized by relative decreases in NADH dehydrogenase and ubiquinol-cytochrome c oxidoreductase activities, which may contribute to the metabolic abnormalities and decreased exercise performance in these patients.
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Tsai, C. S. "Nitroreductase activity of heart lipoamide dehydrogenase." Biochemical Journal 242, no. 2 (March 1, 1987): 447–52. http://dx.doi.org/10.1042/bj2420447.
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A novel reaction catalysed by lipoamide dehydrogenase is described. In the presence of NADH, lipoamide dehydrogenase reduces the nitro group of 4-nitropyridine and 4-nitropyridine N-oxide. The elution profiles from a DEAE-cellulose column for the dehydrogenase and nitroreductase activities are identical. Chemical modifications of critical amino acid residues suggest that the two activities share a common catalytic domain. Nitro reduction catalysed by lipoamide dehydrogenase was monitored spectrophotometrically and chromatographically. The major product from the enzymic reduction of 4-nitropyridine was isolated and characterized structurally as NN-bis(pyridinyl)hydroxylamine, which is formed presumably via 4-hydroxyaminopyridine in a four-electron redox reaction.
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Lopez de Felipe, Felix, Michiel Kleerebezem, Willem M. de Vos, and Jeroen Hugenholtz. "Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase." Journal of Bacteriology 180, no. 15 (August 1, 1998): 3804–8. http://dx.doi.org/10.1128/jb.180.15.3804-3808.1998.
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ABSTRACT NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2gene, which encodes the H2O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD+ratios in the cells.
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Cheema-Dhadli, S., F. A. Halperin, K. Sonnenberg, V. MacMillan, and M. L. Halperin. "Regulation of ethanol metabolism in the rat." Biochemistry and Cell Biology 65, no. 5 (May 1, 1987): 458–66. http://dx.doi.org/10.1139/o87-059.
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The purpose of these experiments was to examine the factors which regulate ethanol metabolism in vivo. Since the major pathway for ethanol removal requires flux through hepatic alcohol dehydrogenase, the activity of this enzyme was measured and found to be 2.9 μmol/(min∙g liver). Ethanol disappearance was linear for over 120 min in vivo and the blood ethanol fell 0.1 mM/min; this is equivalent to removing 20 μmol ethanol/min and would require that flux through alcohol dehydrogenase be about 60% of its measured maximum velocity. To test whether ethanol metabolism was limited by the rate of removal of one of the end products (NADH) of alcohol dehydrogenase, fluoropyruvate was infused to reoxidize hepatic NADH and to prevent NADH generation via flux through pyruvate dehydrogenase. There was no change in the rate of ethanol clearance when fluoropyruvate was metabolized. Furthermore, enhancing endogenous hepatic NADH oxidation by increasing the rate of urea synthesis (converting ammonium bicarbonate to urea) did not augment the steady-state rate of ethanol oxidation. Hence, transport of cytoplasmic reducing power from NADH into the mitochondria was not rate limiting for ethanol oxidation. In contrast, ethanol oxidation at the earliest time periods could be augmented by increasing hepatic urea synthesis.
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Soloveva, Ekaterina R., O. V. Karaseva, M. F. Vasileva, S. V. Petrichuk, I. V. Samokhina, and K. E. Khmel’nitskiy. "THE EFFECT OF MICROWAVES OF A DECIMETER RANGE ON THE FUNCTIONAL ACTIVITY OF MITOCHONDRIA IN DESTRUCTIVE APPENDICITIS IN CHILDREN." Russian Journal of Pediatric Surgery 22, no. 2 (June 9, 2018): 72–77. http://dx.doi.org/10.18821/1560-9510-2018-22-2-72-77.
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Introduction. The article assessed the effectiveness of decimeter wave therapy (DWT) in the postoperative period after laparoscopic appendectomy for destructive appendicitis in children. Authors relate positive clinical effects with the activation of mitochondrial energy metabolism. Material and methods. The study included 132 patients (46 destructive appendicitis cases, 86 h appendicular peritonitis patients). Among them, there were 75 (56.8 percent) boys and 57 (43.2 percent) girls aged of from 3 to 17 years (mean age of 10.7 ± 3.07 years). Patients of the main group received DWT procedures with the use of the apparatus DMV-02 “Solnyshko” starting from the 1st day after the surgery. Patients from the reference group received no physiotherapy treatment. The activity of dehydrogenases of lymphocytes (succinic dehydrogenase (SDH) and NADH-dehydrogenase) in patients from the main group and the comparison group were determined by means of cytomorphological method with the use a hardware-software visualization complex “Videotest” and the “Morphology 5.2” (Russia), at the 1st, 3rd and 5th day of the postoperative period. Results. Under the influence of DWT in the postoperative period the frequency of intestinal insufficiency syndrome, systemic inflammatory response syndrome, infiltrative adhesions was established to decline. There was noted a gain in the activity of mitochondrial enzymes, reflecting the first and second stages of the respiratory chain (SDH and NADH-dehydrogenase). In children with destructive appendicitis against the background of DWT, the normalization of the activity of LDH in moderate activation of NADH-dehydrogenase, and in appendicular peritonitis cases - the significant elevation in LDH activity of lymphocytes and activation of NADH-dehydrogenase occurs. In patients from the main group, the absolute number of peripheral blood lymphocytes normalized Conclusion. Under the influence of DWT there was noted the activation of enzymes of mitochondria that provides a mild course of the postoperative period.
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Kim, Youngnyun, L. O. Ingram, and K. T. Shanmugam. "Dihydrolipoamide Dehydrogenase Mutation Alters the NADH Sensitivity of Pyruvate Dehydrogenase Complex of Escherichia coli K-12." Journal of Bacteriology 190, no. 11 (March 28, 2008): 3851–58. http://dx.doi.org/10.1128/jb.00104-08.
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ABSTRACT Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher Ki for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.
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Schempp, H., H. Ulrich, and E. F. Elstner. "Stereospecific Reduction of /R(+)-Thioctic Acid by Porcine Heart Lipoamide Dehydrogenase/Diaphorase." Zeitschrift für Naturforschung C 49, no. 9-10 (October 1, 1994): 691–92. http://dx.doi.org/10.1515/znc-1994-9-1023.
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Abstract R(+)-thioctic acid is the naturally occurring cofactor in α-ketoacid dehydrogenases. We show both photo metrically by NADH + H+ oxidation and by HPLC product analysis that this enantiomer is rapidly reduced by NADH + H+ catalyzed by porcine heart lipoamide dehydrogenase/diaphorase. The racemate exhibits approxi mately 40% activity as compared to the R (+) form while the S (-) enantiomer photometrically shows little activity and yields no detectable reduced lipoic acid.
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Bakker, Barbara M., Christoffer Bro, Peter Kötter, Marijke A. H. Luttik, Johannes P. van Dijken, and Jack T. Pronk. "The Mitochondrial Alcohol Dehydrogenase Adh3p Is Involved in a Redox Shuttle in Saccharomyces cerevisiae." Journal of Bacteriology 182, no. 17 (September 1, 2000): 4730–37. http://dx.doi.org/10.1128/jb.182.17.4730-4737.2000.
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ABSTRACT NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly, NDI1is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogenases. Deletion of ADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures. Also, an ndi1Δ mutant was capable of respiratory growth under these conditions. However, when both ADH3 and NDI1 were deleted, metabolism became respirofermentative, indicating that the ethanol-acetaldehyde shuttle is essential for respiratory growth of the ndi1Δ mutant. In anaerobic batch cultures, the maximum specific growth rate of the adh3Δ mutant (0.22 h−1) was substantially reduced compared to that of the wild-type strain (0.33 h−1). This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions. Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in theadh3Δ ndi1Δ mutant, contributing to residual redox-shuttle activity in this strain.
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Reed, David W., Jack Millstein, and Patricia L. Hartzell. "H2O2-Forming NADH Oxidase with Diaphorase (Cytochrome) Activity from Archaeoglobus fulgidus." Journal of Bacteriology 183, no. 24 (December 15, 2001): 7007–16. http://dx.doi.org/10.1128/jb.183.24.7007-7016.2001.
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ABSTRACT An enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from the hyperthermophilic sulfate-reducing anaerobeArchaeoglobus fulgidus. N-terminal sequence of the protein indicates that it is coded for by open reading frame AF0395 in theA. fulgidus genome. The gene AF0395 was cloned and its product was purified from Escherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 47 kDa, requires flavin adenine dinucleotide for activity, has NADH-specific activity, and is thermostable. Hydrogen peroxide is the product of bivalent oxygen reduction by rNoxA2 with NADH. The rNoxA2 is an oxidase with diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochrome c. During purification NoxA2 remains associated with the enzyme responsible for d-lactate oxidation, thed-lactate dehydrogenase (Dld), and the genes encoding NoxA2 and Dld are in the same transcription unit. Together these results suggest that NADH oxidase may be involved in electron transfer reactions resulting in sulfate respiration.
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Sales, Cristina R. G., Anabela Bernardes da Silva, and Elizabete Carmo-Silva. "Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays." Journal of Experimental Botany 71, no. 18 (June 30, 2020): 5302–12. http://dx.doi.org/10.1093/jxb/eraa289.
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Abstract Rubisco is central to carbon assimilation, and efforts to improve the efficiency and sustainability of crop production have spurred interest in phenotyping Rubisco activity. We tested the hypothesis that microtiter plate-based methods provide comparable results to those obtained with the radiometric assay that measures the incorporation of 14CO2 into 3-phosphoglycerate (3-PGA). Three NADH-linked assays were tested that use alternative coupling enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycerolphosphate dehydrogenase (GlyPDH); phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH); and pyruvate kinase (PK) and lactate dehydrogenase (LDH). To date there has been no thorough evaluation of their reliability by comparison with the 14C-based method. The three NADH-linked assays were used in parallel to estimate (i) the 3-PGA concentration–response curve of NADH oxidation, (ii) the Michaelis–Menten constant for ribulose-1,5-bisphosphate, (iii) fully active and inhibited Rubisco activities, and (iv) Rubisco initial and total activities in fully illuminated and shaded leaves. All three methods correlated strongly with the 14C-based method, and the PK–LDH method showed a strong correlation and was the cheapest method. PEPC–MDH would be a suitable option for situations in which ADP/ATP might interfere with the assay. GAPDH–GlyPDH proved more laborious than the other methods. Thus, we recommend the PK–LDH method as a reliable, cheaper, and higher throughput method to phenotype Rubisco activity for crop improvement efforts.
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Pearson, J. K., and D. W. Sickles. "Enzyme activity changes in rat soleus motoneurons and muscle after synergist ablation." Journal of Applied Physiology 63, no. 6 (December 1, 1987): 2301–8. http://dx.doi.org/10.1152/jappl.1987.63.6.2301.
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Quantitative enzyme histochemical methods have been used to determine the effect of ablation of synergists on the oxidative metabolism of the alpha-motoneurons and muscle fibers of the rat soleus. Sixty days postablation, the NADH-tetrazolium reductase (NADH-TR) activity of soleus motoneurons decreased 12.5% from 0.327 +/- 0.005 (mean +/- SE; optical density units) to 0.286 +/- 0.007. In the muscle fibers, the alpha-glycerophosphate dehydrogenase activity (glycolytic enzyme) decreased from 0.114 +/- 0.010 to 0.074 +/- 0.009, a change of 35.1%, and the NADH-TR activity decreased 21.2% from 0.348 +/- 0.018 to 0.274 +/- 0.017. In both the motoneurons and the muscle fibers, the decrease was nonspecific for all cells, although a greater effect on the cells with higher enzyme activity was observed. The decreased NADH-TR activity represents a shift in the oxidative profile of the motoneurons and muscle fibers, indicating a decreased ability to use oxidative metabolism for periods of short-term high-energy demands. Furthermore, the parallel decrease in muscle fibers and motoneurons with high NADH-TR activity (fast-twitch oxidative-glycolytic fibers and presumably also motoneurons) demonstrates the tight correlation of the NADH-TR activity between these parts of the motor unit in both control and synergist-ablated muscles.
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Huo, Heyu, Guangxiao Yao, and Shizhen Wang. "Economy Assessment for the Chiral Amine Production with Comparison of Reductive Amination and Transamination Routes by Multi-Enzyme System." Catalysts 10, no. 12 (December 11, 2020): 1451. http://dx.doi.org/10.3390/catal10121451.
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Chiral amines are key building blocks for pharmaceuticals. Economic assessment of commercial potential of bioprocesses is needed for guiding research. Biosynthesis of (S)-α-methylbenzylamine (MBA) was selected as case study. For transamination route, transaminase coupled with glucose dehydrogenase and lactate dehydrogenase catalyzed the reaction with NADH (Nicotinamide adenine dinucleotide) regeneration. Amine dehydrogenase coupled with NADH oxidase, which catalyzed the reductive amination process. Comparison of biosynthesis cost by reductive amination and transamination routes was carried out. Economic assessment based on the framework of cost analysis and preliminary process information revealed that cost is greatly dependent on enzyme price. The results indicated that enhancing the activity of amine dehydrogenase by 4–5 folds can drop the unit price of reductive amination to $0.5–0.6/g, which make it competitive with transamination route.
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Кислова, О. В. "ВПЛИВ ЗАМІЩЕННОГО НІКОТИНАМІДУ ТА ЙОГО МОЖЛИВИХ МЕТАБОЛІТІВ НА АКТИВНІСТЬ ФЕРМЕНТІВ ОБМІНУ ЕТАНОЛУ." Bulletin of the Kyiv National University of Technologies and Design. Technical Science Series 144, no. 2 (October 14, 2020): 98–104. http://dx.doi.org/10.30857/1813-6796.2020.2.10.
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To study the influence of N-phenyl-N-(1-cyclopropylethyl)nicotinamide and its possible metabolites: hydrochlorides of N-(1-cyclopropylethyl)amine and N-phenyl-N-(1-cyclopropylethyl)amine - on the activity of main ethanol oxidation enzymes in vitro and kinetic nature of their interaction. The studies were carried out using alcohol dehydrogenase and aldehyde dehydrogenase of rat liver subcellular fractions, which were obtained by differential centrifugation. The enzyme activity was determined spectrophotometrically. The kinetic nature of alcohol dehydrogenase and isozyme form of aldehyde dehydrogenase interaction with substituted nicotinamide was investigated in the concentration range of 25-100 μM. The research results were processed by the Lineweaver-Burk method. Studies have shown that N-phenyl-N-(1-cyclopropylethyl)nicotinamide is able to reduce the rate of the reverse alcohol dehydrogenase reaction of acetaldehyde reduction to ethanol in the presence of NADH by 46% with an inhibition constant 53 μM. The activity of soluble mitochondrial aldehyde dehydrogenase was suppressed by 50% with an inhibition constant 108 μM. The kinetic nature of the substituted nicotinamide interaction with enzymes at saturating concentrations of the reaction cofactors NADH and NAD+ is quite complex. Allosteric effects can play a significant role in enzymatic activity. Possible metabolites of the compound - hydrochlorides of N-(1-cyclopropylethyl)- and N-phenyl-N-(1-cyclopropylethyl)amine – didn`t significantly influence on ethanol metabolism enzymes activity. A new inhibitor of the rate of the reverse alcohol dehydrogenase reaction and the activity of soluble mitochondrial isozyme form of aldehyde dehydrogenase, which lead to the accumulation of acetaldehyde in the body, has been discovered. N-phenyl-N-(1-cyclopropylethyl)nicotinamide can be used as a potential antialcohol sensitizing drug after research in vivo.
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Singh, Ranji, Ryan J. Mailloux, Simone Puiseux-Dao, and Vasu D. Appanna. "Oxidative Stress Evokes a Metabolic Adaptation That Favors Increased NADPH Synthesis and Decreased NADH Production in Pseudomonas fluorescens." Journal of Bacteriology 189, no. 18 (June 15, 2007): 6665–75. http://dx.doi.org/10.1128/jb.00555-07.
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ABSTRACT The fate of all aerobic organisms is dependent on the varying intracellular concentrations of NADH and NADPH. The former is the primary ingredient that fuels ATP production via oxidative phosphorylation, while the latter helps maintain the reductive environment necessary for this process and other cellular activities. In this study we demonstrate a metabolic network promoting NADPH production and limiting NADH synthesis as a consequence of an oxidative insult. The activity and expression of glucose-6-phosphate dehydrogenase, malic enzyme, and NADP+-isocitrate dehydrogenase, the main generators of NADPH, were markedly increased during oxidative challenge. On the other hand, numerous tricarboxylic acid cycle enzymes that supply the bulk of intracellular NADH were significantly downregulated. These metabolic pathways were further modulated by NAD+ kinase (NADK) and NADP+ phosphatase (NADPase), enzymes known to regulate the levels of NAD+ and NADP+. While in menadione-challenged cells, the former enzyme was upregulated, the phosphatase activity was markedly increased in control cells. Thus, NADK and NADPase play a pivotal role in controlling the cross talk between metabolic networks that produce NADH and NADPH and are integral components of the mechanism involved in fending off oxidative stress.
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Sangiorgi, S., M. Mochi, R. Riva, P. Cortelli, L. Monari, G. Pierangeli, and P. Montagna. "Abnormal Platelet Mitochondrial Function in Patients Affected by Migraine With and Without Aura." Cephalalgia 14, no. 1 (February 1994): 21–23. http://dx.doi.org/10.1046/j.1468-2982.1994.1401021.x.
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To investigate energy metabolism in migraine, we determined platelet mitochondrial enzyme activities in 40 patients with migraine with aura and in 40 patients with migraine without aura during attack-free intervals and in 24 healthy control subjects. NADH-dehydrogenase, citrate synthase and cytochrome-c-oxidase activities in both patient groups were significantly lower than in controls ( p < 0.01), while NADH-cytochrome-c-reductase activity was reduced only in migraine with aura ( p < 0.01). No alteration in succinate-dehydrogenase was observed. Monoamine-oxidase activity differed between sexes (p < 0.05) but within each sex group no difference was observed between patients and controls. We hypothesize that the defect in mitochondrial enzymes observed indicates a systemic impairment of mitochondrial function in migraine patients.
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Jensen, Manfred, Guido B. Feige, and Anna Waterkotte. "Mannitol-1-Phosphate Dehydrogenase in Pseudevernia Furfuracea." Lichenologist 23, no. 2 (April 1991): 187–96. http://dx.doi.org/10.1017/s0024282991000336.
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AbstractMannitol-1-phosphate dehydrogenase [EC 1.1.1.17] activity was demonstrated in extracts of Pseudevernia furfuracea and Hypogymnia physodes. The reaction was found to be highly substrate specific for fructose-6-phosphate/NADH or mannitol-1-phosphate/NAD+. The pH optimum for fructose-6-phosphate reduction was 7.1, and apparent Km values were 1.2 mM for fructose-6-phosphate and 20 μM for NADH. The reaction did not require Mg++ or Ca++. For conversion of mannitol-1-phosphate into mannitol, the occurrence of mannitol-1-phosphatase in Pseudevernia furfuracea is postulated.
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Ke, Dangyang, Elhadi Yahia, Betty Hess, Lili Zhou, and Adel A. Kader. "Regulation of Fermentative Metabolism in Avocado Fruit under Oxygen and Carbon Dioxide Stresses." Journal of the American Society for Horticultural Science 120, no. 3 (May 1995): 481–90. http://dx.doi.org/10.21273/jashs.120.3.481.
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`Hass' avocado (Persea americana Mill.) fruit were kept in air, 0.25% O2 (balance N2), 20 % O2 + 80% CO2, or 0.25% O2 + 80% CO2 (balance N2) at 20C for up to 3 days to study the regulation of fermentative metabolism. The 0.25% 02 and 0.25% 02 + 80% CO2 treatments caused accumulations of acetaldehyde and ethanol and increased NADH concentration, but decreased NAD level. The 20% O2 + 80% CO2 treatment slightly increased acetaldehyde and ethanol concentrations without significant effects on NADH and NAD levels. Lactate accumulated in avocadoes kept in 0.25 % 02. The 80% CO, (added to 0.25% O2) did not increase lactate concentration and negated the 0.25% O2-induced lactate accumulation. Activities of PDC and LDH were slightly enhanced and a new isozyme of ADH was induced by 0.25% O2, 20% O2 + 80% CO2, or 0.25 % O2 + 80% CO2; these treatments partly reduced the overall activity of the PDH complex. Fermentative metabolism can be regulated by changes in levels of PDC, ADH, LDH, and PDH enzymes and/or by metabolic control of the functions of these enzymes through changes in pH, ATP, pyruvate, acetaldehyde, NADH, or NAD. Chemical names used: alcohol dehydrogenase (ADH), adenosine triphosphate (ATP), lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD), reduced NAD (NADH), pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH).
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DUARTE, Margarida, Markus PETERS, Ulrich SCHULTE, and Arnaldo VIDEIRA. "The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria." Biochemical Journal 371, no. 3 (May 1, 2003): 1005–11. http://dx.doi.org/10.1042/bj20021374.
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An open reading frame homologous with genes of non-proton-pumping NADH dehydrogenases was identified in the genome of Neurospora crassa. The 57 kDa NADH:ubiquinone oxidoreductase acts as internal (alternative) respiratory NADH dehydrogenase (NDI1) in the fungal mitochondria. The precursor polypeptide includes a pre-sequence of 31 amino acids, and the mature enzyme comprises one FAD molecule as a prosthetic group. It catalyses specifically the oxidation of NADH. Western blot analysis of fungal mitochondria fractionated with digitonin indicated that the protein is located at the inner face of the inner membrane of the organelle (internal enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. The respiratory activity of mitochondria from the resulting null-mutant ndi1 is almost fully inhibited by rotenone, an inhibitor of the proton-pumping complex I, when matrix-generated NADH is used as substrate. Although no effects of the NDI1 defect on vegetative growth and sexual differentiation were observed, the germination of both sexual and asexual ndi1 mutant spores is significantly delayed. Crosses between the ndi1 mutant strain and complex I-deficient mutants yielded no viable double mutants. Our data indicate: (i) that NDI1 represents the sole internal alternative NADH dehydrogenase of Neurospora mitochondria; (ii) that NDI1 and complex I are functionally complementary to each other; and (iii) that NDI1 is specially needed during spore germination.
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BOWKER-KINLEY, Melissa M., I. Wilhelmina DAVIS, Pengfei WU, A. Robert HARRIS, and M. Kirill POPOV. "Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex." Biochemical Journal 329, no. 1 (January 1, 1998): 191–96. http://dx.doi.org/10.1042/bj3290191.
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Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50 nmol/min per mg for PDK2 to 1250 nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.
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Dickinson, F. M., and G. W. Haywood. "The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae." Biochemical Journal 247, no. 2 (October 15, 1987): 377–84. http://dx.doi.org/10.1042/bj2470377.
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The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process. Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4%) in the complete absence of K+. The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH. Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex. The variation of activity of the enzyme with pH was studied. An enzyme group with pKa approx. 7.1 apparently promotes enzyme activity when in its alkaline form.
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Topham, R., M. Goger, K. Pearce, and P. Schultz. "The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates." Biochemical Journal 261, no. 1 (July 1, 1989): 137–43. http://dx.doi.org/10.1042/bj2610137.
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Considerable evidence suggests that the release of iron from ferritin is a reductive process. A role in this process has been proposed for two hepatic enzymes, namely xanthine oxidoreductase and an NADH oxidoreductase. The abilities of xanthine and NADH to serve as a source of reducing power for the enzyme-mediated release of ferritin iron (ferrireductase activity) were compared with turkey liver and rat liver homogenates. The maximal velocity (Vmax.) for the reaction with NADH was 50 times greater than with xanthine; however, the substrate concentration required to achieve half-maximal velocity (Km) was 1000 times less with xanthine than with NADH. NADPH could be substituted for NADH with little loss in activity. Dicoumarol did not inhibit the reaction with NADH or NADPH, demonstrating that the ferrireductase activity with those substrates was not the result of the liver enzyme ‘DT-diaphorase’ [NAD(P)H dehydrogenase (quinone)]. A flavin nucleotide was required for ferrireductase activity with rat and turkey liver cytosol when xanthine, NADH or NADPH was used as the reducing substrate. FMN yielded twice the activity with NADH or NADPH, whereas FAD was twice as effective with xanthine as substrate. Kinetic comparisons, differences in lability and partial chromatographic resolution of the ferrireductase activities with the two types of reducing substrates strongly indicate that the ferrireductase activities with xanthine and NADH are catalysed by separate enzyme systems contained in liver cytosol. Complete inhibition by allopurinol of the ferrireductase activity endogenous to undialysed liver cytosol preparations and the ability of xanthine to restore equivalent activity to dialysed preparations indicate that the source of reducing power for the endogenous activity is xanthine. These studies suggest that xanthine, NADH or NADPH can serve as a source of reducing power for the enzyme-mediated reduction of ferritin iron, with a flavin nucleotide serving as the shuttle of electrons from the enzymes to the ferritin iron.
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Kanbe, Chiyuki, and Kinji Uchida. "NADH Dehydrogenase Activity ofPediococcus halophilusas a Factor Determining its Reducing Force." Agricultural and Biological Chemistry 51, no. 2 (February 1987): 507–14. http://dx.doi.org/10.1080/00021369.1987.10868072.
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Popov, Kirill M., Natalia Y. Kedishvili, and Robert A. Harris. "Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1119, no. 1 (February 1992): 69–73. http://dx.doi.org/10.1016/0167-4838(92)90236-7.
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Wharton, M., D. L. Granger, and D. T. Durack. "Mitochondrial iron loss from leukemia cells injured by macrophages. A possible mechanism for electron transport chain defects." Journal of Immunology 141, no. 4 (August 15, 1988): 1311–17. http://dx.doi.org/10.4049/jimmunol.141.4.1311.
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Abstract Activated macrophages inhibit replication of murine lymphoblastic leukemia L1210 cells without lysis. This inhibition of replication is associated with abnormalities of mitochondrial electron transport at the level of NADH dehydrogenase (NADH-DH) and succinate dehydrogenase (SDH). The mechanism of inhibition is unknown, although it has been demonstrated that as NADH-DH and SDH activity is lost, iron is released from cells. Because both NADH-DH and SDH contain numerous iron-sulfur clusters, damage to these structures may be one result of injury by activated macrophages. L1210 cells were labeled with 55Fe and co-cultivated with activated murine peritoneal macrophages (injured L1210 cells). At 48 h, injured L1210 cells had released 83 +/- 8% (mean +/- SEM of 55Fe activity into the media, compared with 25 +/- 4% release from control and 37 +/- 7% from nondividing mitomycin C-treated control cells. All cells were greater than 90% viable. These differences were also reflected in the iron content of the cells. Mitochondria were then separated by centrifugation after cell disruption and 55Fe activity was found to be similarly decreased in both mitochondrial and nonmitochondrial fractions of injured L1210 cells. To further characterize the changes in mitochondrial iron content, mitochondrial proteins from injured and control L1210 cells were separated by IEF and 55Fe activity of gel slices was determined. There was selective loss of 55Fe activity in the area of the gel corresponding to SDH and NADH-DH, suggesting that iron loss from iron-sulfur clusters may occur in L1210 cells injured by activated macrophages. Iron uptake into L1210 cells after removal from macrophages showed a rapid large influx of radioactive iron. L1210 cells in contact with macrophages appear to develop an iron-depleted state, which is dependent on the continued presence of macrophages.
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Alissandratos, Apostolos, Hye-Kyung Kim, Hayden Matthews, James E. Hennessy, Amy Philbrook, and Christopher J. Easton. "Clostridium carboxidivorans Strain P7T Recombinant Formate Dehydrogenase Catalyzes Reduction of CO2to Formate." Applied and Environmental Microbiology 79, no. 2 (November 9, 2012): 741–44. http://dx.doi.org/10.1128/aem.02886-12.
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ABSTRACTRecombinant formate dehydrogenase from the acetogenClostridium carboxidivoransstrain P7T, expressed inEscherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions.
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González-Pajuelo, María, Isabelle Meynial-Salles, Filipa Mendes, Philippe Soucaille, and Isabel Vasconcelos. "Microbial Conversion of Glycerol to 1,3-Propanediol: Physiological Comparison of a Natural Producer, Clostridium butyricum VPI 3266, and an Engineered Strain, Clostridium acetobutylicum DG1(pSPD5)." Applied and Environmental Microbiology 72, no. 1 (January 2006): 96–101. http://dx.doi.org/10.1128/aem.72.1.96-101.2006.
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ABSTRACT Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same “global behavior” was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH2 flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD+ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.
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Sheeran, Freya L., Julie Angerosa, Norman Y. Liaw, Michael M. Cheung, and Salvatore Pepe. "Adaptations in Protein Expression and Regulated Activity of Pyruvate Dehydrogenase Multienzyme Complex in Human Systolic Heart Failure." Oxidative Medicine and Cellular Longevity 2019 (February 7, 2019): 1–11. http://dx.doi.org/10.1155/2019/4532592.
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Pyruvate dehydrogenase (PDH) complex, a multienzyme complex at the nexus of glycolytic and Krebs cycles, provides acetyl-CoA to the Krebs cycle and NADH to complex I thus supporting a critical role in mitochondrial energy production and cellular survival. PDH activity is regulated by pyruvate dehydrogenase phosphatases (PDP1, PDP2), pyruvate dehydrogenase kinases (PDK 1-4), and mitochondrial pyruvate carriers (MPC1, MPC2). As NADH-dependent oxidative phosphorylation is diminished in systolic heart failure, we tested whether the left ventricular myocardium (LV) from end-stage systolic adult heart failure patients (n=26) exhibits altered expression of PDH complex subunits, PDK, MPC, PDP, and PDH complex activity, compared to LV from nonfailing donor hearts (n=21). Compared to nonfailing LV, PDH activity and relative expression levels of E2, E3bp, E1α, and E1βsubunits were greater in LV failure. PDK4, MPC1, and MPC2 expressions were decreased in failing LV, whereas PDP1, PDP2, PDK1, and PDK2 expressions did not differ between nonfailing and failing LV. In order to examine PDK4 further, donor human LV cardiomyocytes were induced in culture to hypertrophy with 0.1 μM angiotensin II and treated with PDK inhibitors (0.2 mM dichloroacetate, or 5 mM pyruvate) or activators (0.6 mM NADH plus 50 μM acetyl CoA). In isolated hypertrophic cardiomyocytesin vitro, PDK activators and inhibitors increased and decreased PDK4, respectively. In conclusion, in end-stage failing hearts, greater expression of PDH proteins and decreased expression of PDK4, MPC1, and MPC2 were evident with higher rates of PDH activity. These adaptations support sustained capacity for PDH to facilitate glucose metabolism in the face of other failing bioenergetic pathways.
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Omar, M. S., and A. M. S. Raoof. "Onchocerca fasciata: histochemical demonstration of succinate and NADH dehydrogenase." Journal of Helminthology 70, no. 1 (March 1996): 47–51. http://dx.doi.org/10.1017/s0022149x00015121.
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AbstractThe activities of selected enzymes of the respiratory chain system in Onchocerca fasciata (Filarioidea: Onchocercidae) have been investigated histochemically. Thus, the localization and distributions of NADH dehydrogenase (EC 1.6.99.3), succinate dehydrogenase (SDH) (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) were investigated in various tissues of the adult female worm by employing MTT, Nitro BT (dehydrogenases) and DAB (cytochrome oxidase). Different tissues varied considerably in their enzymatic activities. The hypodermis and reproductive tissues showed strong and identical localization of NADH and SDH dehydrogenase activities reflecting high metabolic rates. Little or no dehydrogenase activities were observed in the intestine and cuticle. In contrast to the two dehyrogenases, no activity was observed for cytochrome oxidase in any of the tissues in adult or embryonic stages of the worm. The significance of these results with respect to the energy metabolism of the worm is discussed. It is suggested that O. fasciata lacks a classical, mammalian-type respiratory pathway and that oxidative phosphorylation is of no importance as an energy generating pathway in this essentially anaerobic parasite.
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Granat, Lucy, Debbra Y. Knorr, Daniel C. Ranson, Emma L. Hamer, Ram Prosad Chakrabarty, Francesca Mattedi, Laura Fort-Aznar, et al. "Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency." PLOS Genetics 19, no. 7 (July 3, 2023): e1010793. http://dx.doi.org/10.1371/journal.pgen.1010793.
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Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.
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CA, Murphy, Large PJ, C. Wadforth, Dack SJ, and Boulton CA. "Strain‐dependent variation in the NADH‐dependent diacetyl reductase activities of larger‐ and alebrewing yeasts." Biotechnology and Applied Biochemistry 23, no. 1 (February 1996): 19–22. http://dx.doi.org/10.1111/j.1470-8744.1996.tb00359.x.
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Significant differences were observed in the zymogram patterns of NAD(+)‐dependent ethanol dehydrogenase and acetoin dehydrogenase activity in seven strains of brewer's yeast examined by non‐denaturing PAGE. Bottom‐fermenting (lager) strains contained quite different activity bands of acetoin dehydrogenase activity compared with top‐fermenting (ale) strains. These differences were confirmed when cell‐free extracts of ale yeasts were heated at 55 degrees C. This destroyed most of the diacetyl reductase activity, while leaving acetaldehyde reductase and other reductase activities unaffected. In contrast, heating cell‐free extracts of lager yeasts at 55 degrees C inactivated diacetyl reductase activity and the other reductase activities at the same rate, and more slowly than with ale strains. Similar distinctions between the two types of yeast could be made by examining the effect of heat on the ratio (activity of the various substrates with NADH as electron donor)/(activity with reduced acetylpyridine‐adenine dinucleotide as electron donor). The data show that the acetoin dehydrogenase/diacetyl reductase enzyme present in ale‐yeast strains differs in mobility and heat‐stability from that of larger strains, and that both can be distinguished from the major alcohol dehydrogenase activity bands.
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Burgess, Shawn C., Katsumi Iizuka, Nam Ho Jeoung, Robert A. Harris, Yoshihiro Kashiwaya, Richard L. Veech, Tatsuya Kitazume, and Kosaku Uyeda. "Carbohydrate-response Element-binding Protein Deletion Alters Substrate Utilization Producing an Energy-deficient Liver." Journal of Biological Chemistry 283, no. 3 (November 27, 2007): 1670–78. http://dx.doi.org/10.1074/jbc.m706540200.
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Livers from mice lacking the carbohydrate-responsive element-binding protein (ChREBP) were compared with wild type (WT) mice to determine the effect of this transcription factor on hepatic energy metabolism. The pyruvate dehydrogenase complex was considerably more active in ChREBP-/- mice because of diminished pyruvate dehydrogenase kinase activity. Greater pyruvate dehydrogenase complex activity caused a stimulation of lactate and pyruvate oxidation, and it significantly impaired fatty acid oxidation in perfused livers from ChREBP-/- mice. This shift in mitochondrial substrate utilization led to a 3-fold reduction of the free cytosolic [NAD+]/[NADH] ratio, a 1.7-fold increase in the free mitochondrial [NAD+]/[NADH] ratio, and a 2-fold decrease in the free cytosolic [ATP]/[ADP][Pi] ratio in the ChREBP-/- liver compared with control. Hepatic pyruvate carboxylase flux was impaired with ChREBP deletion secondary to decreased fatty acid oxidation, increased pyruvate oxidation, and limited pyruvate availability because of reduced activity of liver pyruvate kinase and malic enzyme, which replenish pyruvate via glycolysis and pyruvate cycling. Overall, the shift from fat utilization to pyruvate and lactate utilization resulted in a decrease in the energy of ATP hydrolysis and a hypo-energetic state in the livers of ChREBP-/- mice.
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Marbaix, Alexandre Y., Georges Chehade, Gaëtane Noël, Pierre Morsomme, Didier Vertommen, Guido T. Bommer, and Emile Van Schaftingen. "Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P)+." Biochemical Journal 476, no. 20 (October 28, 2019): 3033–52. http://dx.doi.org/10.1042/bcj20190602.
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Abstract 6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5′-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this ‘side-activity’ is conserved. Remarkably, ‘pyridoxamine-phosphate oxidase-related proteins’ (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5′-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).
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Kalnenieks, Uldis, Malda M. Toma, Nina Galinina, and Robert K. Poole. "The paradoxical cyanide-stimulated respiration of Zymomonas mobilis: cyanide sensitivity of alcohol dehydrogenase (ADH II)." Microbiology 149, no. 7 (July 1, 2003): 1739–44. http://dx.doi.org/10.1099/mic.0.26073-0.
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The respiratory inhibitor cyanide stimulates growth of the ethanologenic bacterium Zymomonas mobilis, perhaps by diverting reducing equivalents from respiration to ethanol synthesis, thereby minimizing accumulation of toxic acetaldehyde. This study sought to identify cyanide-sensitive components of respiration. In aerobically grown, permeabilized Z. mobilis cells, addition of 200 μM cyanide caused gradual inhibition of ADH II, the iron-containing alcohol dehydrogenase isoenzyme, which, in aerobic cultures, might be oxidizing ethanol and supplying NADH to the respiratory chain. In membrane preparations, NADH oxidase was inhibited more rapidly, but to a lesser extent, than ADH II. The time-course of inhibition of whole-cell respiration resembled that of NADH oxidase, yet the inhibition was almost complete, and was accompanied by an increase of intracellular NADH concentration. Cyanide did not significantly affect the activity of ADH I, the zinc-containing alcohol dehydrogenase isoenzyme. When an aerobic batch culture was grown in the presence of 200 μM cyanide, cyanide-resistant ADH II activity was observed, its appearance correlating with the onset of respiration. It is concluded that the membrane-associated respiratory chain, but not ADH II, is responsible for the whole-cell cyanide sensitivity, while the cyanide-resistant ADH II is needed for respiration in the presence of cyanide, and represents an adaptive response of Z. mobilis to cyanide, analogous to the induction of alternative terminal oxidases in other bacteria.
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Wang, Yaping, Yanhong Peng, Xiaoyan Liu, Ronghua Zhou, Xianqing Liao, Yong Min, Yong Hu, Ying Wang, and Ben Rao. "Efficient 2,3-Butanediol/Acetoin Production Using Whole-Cell Biocatalyst with a New Nadh/Nad(+) Regeneration System." Catalysts 11, no. 12 (November 23, 2021): 1422. http://dx.doi.org/10.3390/catal11121422.
Full textAbstract:
An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.
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Ogura, Masato, Junko Yamaki, Miwako K. Homma, and Yoshimi Homma. "Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components." Biochemical Journal 447, no. 2 (September 26, 2012): 281–89. http://dx.doi.org/10.1042/bj20120509.
Full textAbstract:
Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.
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Mankovska, I. M., O. O. Gonchar, and L. V. Bratus. "THE EFFECT OF MEXIDOL ON GLUTATHIONE SYSTEM IN RAT BRAIN UNDER MODELING OF PARKINSON’S DESEASE." Fiziolohichnyĭ zhurnal 68, no. 1 (January 18, 2022): 13–19. http://dx.doi.org/10.15407/fz68.01.013.
Full textAbstract:
We studied the effects of mexidol (3-oxy-6-methyl-2-ethylpiridine succinate) on the antioxidant glutathione system in rat brain mitochondria in experimental Parkinson’s disease induced by rotenone administration. Wistar rats were divided into the following groups of 6 in each: I - intact rats (control); II - rotenone (3 mg/kg per day) was injected subcutaneously for 2 weeks; III - after rotenone intoxication, mexidol (50 mg/kg per day) was injected intraperitoneally for 2 weeks. In the suspension of brain mitochondria, the activity of NADH dehydrogenase (complex I of the mitochondrial respiratory chain), content of the active products of 2-thiobarbituric acid (TBA-AP), the reduced (GSH) and oxidized (GSSG) glutathione amounts, the activity of glutathione-dependent enzymes: glutathione peroxidase (GP) and glutathione reductase (GR) as well as NADH+-isocitrate-dehydrogenase activity (NADPH+- ICDH) were measured. The activity and protein expression of MnSOD and GP in rat brain mitochondria were estimated. Treatment of rats with mexidol led to a weakening of oxidative processes in brain mitochondria in comparison with rats exposed to rotenone intoxication. It was shown that intraperitoneal injections of mexidol led to a decrease in the TBA-AP and in the GSSG content and to an increase in GSH/GSSG ratio in comparison with rotenone intoxication. It was also registered an increase in the activity of NADH-dehydrogenase. Such changes indicated a weakening of the mitochondrial oxidative processes intensity. Treatment of rats with mexidol promoted an increase in GSH content, GR and NADPH+-ICDH activities in brain mitochondria in comparison with rotenone administration. Treatment with mexidol resulted to an increased activity and protein expression of GP and MnSOD. We conclude that mexidol reduced the rotenone-induced damage of rat brain mitochondria increasing the action of glutathione-dependent and NADPH+-generating enzymes.
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Boyer, B., and R. Odessey. "Quantitative control analysis of branched-chain 2-oxo acid dehydrogenase complex activity by feedback inhibition." Biochemical Journal 271, no. 2 (October 15, 1990): 523–28. http://dx.doi.org/10.1042/bj2710523.
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The potential for branched-chain 2-oxo acid dehydrogenase complex (BCOADC) activity to be controlled by feedback inhibition was investigated by calculating the Elasticity Coefficients for several feedback inhibitors. We suggest that feedback inhibition is a quantitatively important regulatory mechanism by which branched-chain 2-oxo acid dehydrogenase activity is regulated. The potential for control of enzyme activity is greater for NADH than for the acyl-CoA products, and suggests that factors that alter the redox potential may physiologically regulate BCOADC activity through a feedback inhibitory mechanism in vivo. Local pH may also be an important regulatory control factor.
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Camacho Carvajal, Margarita M., André H. M. Wijfjes, Ine H. M. Mulders, Ben J. J. Lugtenberg, and Guido V. Bloemberg. "Characterization of NADH Dehydrogenases of Pseudomonas fluorescens WCS365 and Their Role in Competitive Root Colonization." Molecular Plant-Microbe Interactions® 15, no. 7 (July 2002): 662–71. http://dx.doi.org/10.1094/mpmi.2002.15.7.662.
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The excellent-root-colonizing Pseudomonas fluorescens WCS365 was selected previously as the parental strain for the isolation of mutants impaired in root colonization. Transposon mutagenesis of WCS365 and testing for root colonization resulted in the isolation of mutant strain PCL1201, which is approximately 100-fold impaired in competitive tomato root colonization. In this manuscript, we provide evidence that shows that the lack of NADH dehydrogenase I, an enzyme of the aerobic respiratory chain encoded by the nuo operon, is responsible for the impaired root-colonization ability of PCL1201. The complete sequence of the nuo operon (ranging from nuoA to nuoN) of P. fluorescens WCS365 was identified, including the promoter region and a transcriptional terminator consensus sequence downstream of nuoN. It was shown biochemically that PCL1201 is lacking NADH dehydrogenase I activity. In addition, the presence and activity of a second NADH dehydrogenase, encoded by the ndh gene, was identified to our knowledge for the first time in the genus Pseudomonas. Since it was assumed that low-oxygen conditions were present in the rhizosphere, we analyzed the activity of the nuo and the ndh promoters at different oxygen tensions. The results showed that both promoters are up-regulated by low concentrations of oxygen and that their levels of expression vary during growth. By using lacZ as a marker, it was shown that both the nuo operon and the ndh gene are expressed in the tomato rhizosphere. In contrast to the nuo mutant PCL1201, an ndh mutant of WCS365 appeared not to be impaired in competitive root tip colonization.
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Marcillat, O., Y. Zhang, and K. J. A. Davies. "Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin." Biochemical Journal 259, no. 1 (April 1, 1989): 181–89. http://dx.doi.org/10.1042/bj2590181.
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The quinonoid anthracycline, doxorubicin (Adriamycin) is a potent anti-neoplastic agent whose clinical use is limited by severe cardiotoxicity. Mitochondrial damage is a major component of this cardiotoxicity, and rival oxidative and non-oxidative mechanisms for inactivation of the electron transport chain have been proposed. Using bovine heart submitochondrial preparations (SMP) we have now found that both oxidative and non-oxidative mechanisms occur in vitro, depending solely on the concentration of doxorubicin employed. Redox cycling of doxorubicin by Complex I of the respiratory chain (which generates doxorubicin semiquinone radicals, O2-, H2O2, and .OH) caused a 70% decrease in the Vmax. for NADH dehydrogenase during 15 min incubation of SMP, and an 80% decrease in NADH oxidase activity after 2 h incubation. This inactivation required only 25-50 microM-doxorubicin and represents true oxidative damage, since both NADH (for doxorubicin redox cycling) and oxygen were obligatory participants. The damage appears localized between the NADH dehydrogenase flavin (site of doxorubicin reduction) and iron-sulphur centre N-1. Succinate dehydrogenase, succinate oxidase, and cytochrome c oxidase activities were strongly inhibited by higher doxorubicin concentrations, but this phenomenon did not involve doxorubicin redox cycling (no NADH or oxygen requirement). Doxorubicin concentrations of 0.5 mM were required for 50% decreases in these activities, except for cytochrome c oxidase which was only 30% inhibited following incubation with even 1.0 mM-doxorubicin. Our results indicate that low concentrations of doxorubicin (50 microM or less) can catalyse a site-specific oxidative damage to the NADH oxidation pathway. In contrast, ten-fold higher doxorubicin concentrations (or more) are required for non-oxidative inactivation of the electron transport chain; probably via binding to cardiolipin and/or generalized membrane chaotropic effects. The development of agents to block doxorubicin toxicity in vivo will clearly require detailed clinical studies of doxorubicin uptake in the heart.
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Hirashima, Y., A. A. Farooqui, and L. A. Horrocks. "Fluorimetric coupled enzyme assay for lysoplasmalogenase activity in liver." Biochemical Journal 260, no. 2 (June 1, 1989): 605–8. http://dx.doi.org/10.1042/bj2600605.
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We developed a continuous spectrofluorimetric assay of lysoplasmalogenase activity with the use of horse liver alcohol dehydrogenase as a coupling enzyme. In this method the disappearance of NADH is measured spectrofluorimetrically. The excitation and emission monochromators were set at 340 and 460 nm respectively. The assay is 10 times as sensitive as the previous u.v. spectrophotometric method. We could detect approx. 0.02 nmol of aldehyde produced/min per ml.