Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits from Neurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits in Neurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.
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PMID:Functional molecular aspects of the NADH dehydrogenases of plant mitochondria. 859 75

This study was undertaken to investigate the effects of growth hormone (GH) on the in vitro maturation of the metabolism of fetal rat islets. For this purpose fetal islets were obtained from 21-day-old fetuses by mild collagenase digestion of the pancreas and cultured in RPMI 1640 supplemented with 10% fetal calf serum. After one day the medium was changed and supplemented with 1% fetal calf serum with or without GH (1 microgram/ml, human recombinant) and the islets cultured for another two days. Islets were then studied with regard to insulin secretion, (pro)insulin and total protein biosynthesis, glucose utilization and oxidation, thymidine incorporation, insulin and DNA contents and the contents of mRNAs for either insulin, adenine nucleotide translocator or cytochrome b. In addition, the activities of glucose phosphorylating enzymes and succinate-cytochrome c reductase were measured. Islets treated with GH showed increased insulin secretion in response to glucose, increased rates of glucose oxidation and utilization, increased thymidine incorporation and increased activities of succinate cytochrome c reductase and glucose phosphorylation at high glucose concentrations. There were, however, no changes in (pro)insulin and total protein biosynthesis, contents of insulin and DNA or the contents of any of the mRNAs. These combined data show that fetal beta-cells are sensitive to growth hormone with respect to glucose metabolism, insulin release and DNA replication. The increased rates of islet glucose phosphorylation may reflect glucokinase activity and explain part of the increased insulin responsiveness to glucose of the fetal rat beta-cell. These observations suggest that GH is of physiological significance for the maturation of the fetal beta-cell.
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PMID:Effects of growth hormone in vitro on the glucose metabolism of fetal rat islet beta-cells. 893 88

A disturbed energy metabolism in pancreatic acinar cells is discussed as factor contributing to the development of acute pancreatitis (AP). In this study, we investigated to what extent the mitochondrial ATP producing capacity is impaired in the pancreatic tissue of rats with experimental AP. For preparation of mitochondria from rat pancreas, routine isolation procedures (tissue homogenization and differential centrifugation) were applied. Mitochondria were isolated from rats with edematous pancreatitis produced by hyperstimulation with caerulein, and from rats with mild necrotizing acute pancreatitis. The latter form of AP was induced by a temporary occlusion of the biliary pancreatic duct accompanied by a simultaneous intravenous injection of caerulein plus secretin and an intraabdominal administration of ethanol. As functional parameters of oxidative phosphorylation, the respiration rate, the mitochondrial membrane potential, and the activity of the complex I of the respiratory chain were determined. Mitochondria from rats with caerulein AP showed an enhanced respiration (61% vs. saline control) and a diminished membrane potential (-17 mV) if respiring with succinate in the non-phosphorylating state. This indicates an increased proton leak across the mitochondrial inner membrane. In the mild necrotizing AP, mitochondria were characterized by a decreased respiration with NAD(+)-linked substrates (-33% vs. sham-operated animals). This inhibition of respiration was confirmed by the reduced activity measured for the NADH-cytochrome c reductase (-32%). In both models of experimental AP the potency of mitochondria to produce ATP was significantly diminished. The stronger impairment of mitochondrial functions were found in the necrotizing form of AP. Reactive oxygen species may lead to the observed alterations--to the enhanced permeability of the mitochondrial inner membrane as well as to the inhibition of the complex I of the respiratory chain.
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PMID:[Modification of energy supply by pancreatic mitochondria in acute experimental pancreatitis]. 922 43

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.
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PMID:A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves. 1212 55

Isosteviol lactone (LAC), a lactone derivative of the diterpenic acid isosteviol (ISO) was evaluated for its effect on the oxidative metabolism of mitochondria isolated from rat liver. In this model, LAC (1 mM) depressed the phosphorylation efficiency, as shown by the decreased respiratory control coefficient (RCC) and ADP/O ratio. LAC (1 mM) inhibited NADH oxidase (45%), succinate oxidase (34%) and promoted low-level inhibitions on succinate dehydrogenase (13%), succinate-cytochrome c oxide-reductase (23%), cytochrome c oxidase (10%), and NADH dehydrogenase (13%). Glutamate dehydrogenase was also a target for LAC, as it was 85% inhibited by 1 mM LAC. Cyclic voltammetry data showed that LAC, as well as ISO, does not undergo redox reactions under current experimental conditions. LAC (0.05-0.75 mM) inhibited the swelling dependent on the glutamate oxidation, 50% of the effect occurring at 0.5 mM LAC. Swelling supported by KNO(3) and valinomycin was also inhibited over all concentrations used of LAC and ISO, the effect being of a lower intensity for LAC, suggesting that the modification of the structure of ISO by lactonization diminished its interaction with the membrane. This could contribute to attenuation of the toxic effects described for ISO on mitochondrial function, such as those on respiratory chain enzymatic complexes and phosphorylating activity.
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PMID:Activity of isosteviol lactone on mitochondrial metabolism. 1269 84

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained approximately 12 per cent of the protein N and approximately 35 per cent of the phospholipide of the whole mitochondria and accounted for approximately 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.
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PMID:Cytochemical studies of mitochondria. II. Enzymes associated with a mitochondrial membrane fraction. 1339 36

The respiration of dark-grown Nicotiana glutinosa L. cells in liquid suspension culture was found to be highly cyanide resistant and salicylhydroxamic acid (SHAM) sensitive, indicative of an active alternative respiratory pathway. This was especially true during the lag and logarithmic phases of the 14-day growth cycle. Mitochondria isolated from logarithmically growing cells exhibited active oxidation of malate, succinate, and exogenous NADH. Oxidation of all three substrates had an optimum pH of 6.5 and all were highly resistant to inhibited by cyanide and sensitive to SHAM. Respiratory control was exhibited by all three substrates but only if SHAM was present to block the alternative pathway and divert electrons to the phosphorylating cytochrome pathway. The cyanide-resistant oxidation of exogenous NADH has previously only been associated with Arum spadix mitochondria. Coemergence during evolution of the alternative respiratory pathway and the exogenous NADH dehydrogenase in plant mitochondria as a possible mechanism for removal of cytoplasmic NADH is proposed. Evidence is presented which suggests that mitochondrial assays should be performed at pH 6.5.
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PMID:Cyanide-Resistant Respiration in Suspension Cultured Cells of Nicotiana glutinosa L. 1666 19

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I-V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD(+)-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast's exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.
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PMID:Mitochondrial function in the yeast form of the pathogenic fungus Paracoccidioides brasiliensis. 1879 87

In mitochondria isolated from the yeast Saccharomyces cerevisiae, under non-phosphorylating conditions, we have previously shown that there is a right of way for electrons coming from the external NADH dehydrogenase, Nde1p. In this work, we show that the electron competition process is identical under more physiological conditions i.e. oxidative phosphorylation. Such a competition generates a priority for cytosolic NADH reoxidation. Furthermore, this electron competition process is associated with an energy wastage (the "active leak") that allows an increase in redox equivalent oxidation when the redox pressure increases. When this redox pressure is decreased, i.e. under phosphorylating conditions, most of this energy wastage is alleviated. By studying mutant strains affected either in respiratory chain supramolecular organization or in electron competition activity, we show that the respiratory chain supramolecular organization is not responsible for the electron competition processes. Moreover, we show two distinct relationships between the respiratory rate and the quinone redox state that seem to indicate two quinone pools that are involved in the electron right of way. Indeed, the more reduced pool would be associated to the electron right of way for the external dehydrogenases whereas the less reduced pool would be associated to the electron right of way for the internal dehydrogenases.
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PMID:Electron competition process in respiratory chain: regulatory mechanisms and physiological functions. 2011 78

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 Tyr(193) of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr(215) 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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PMID:Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components. 2282 20


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