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)

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to alpha (54 kDa), beta (43 kDa), gamma (19 kDa), and delta (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric alphabetagammadelta structure. Functional analysis of each subunit revealed that the beta subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the alpha subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.
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PMID:Gene and primary structures of dye-linked L-proline dehydrogenase from the hyperthermophilic archaeon Thermococcus profundus show the presence of a novel heterotetrameric amino acid dehydrogenase complex. 1506 76

We previously showed that pancreatic beta-cells express a neuronal isoform of nitric oxide synthase (nNOS) that controls insulin secretion by exerting two enzymatic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now bring evidence that two inhibitors of nNOS, N-omega-nitro-l-arginine methyl ester (l-NAME) and 7-nitroindazole (7-NI), increase glucose-induced insulin secretion but affect beta-cell function differently. In the presence of l-NAME, insulin response is monophasic, whereas 7-NI preserves the normal biphasic secretory pattern. In addition, the alterations of beta-cell functional response induced by the inhibitors also differ by their sensitivity to a substitutive treatment with sodium nitroprusside, a chemical NO donor. These differences are probably related to the nature of the two inhibitors. Indeed, using low-temperature SDS-PAGE and real-time analysis of nNOS dimerization by surface plasmon resonance, we could show that 7-NI, which competes with arginine and tetrahydrobiopterin (BH(4)), an essential cofactor for nNOS dimer formation, inhibits dimerization of the enzyme, whereas the substrate-based inhibitor l-NAME stabilizes the homodimeric state of nNOS. The latter effect could be reproduced by the two endogenous inhibitors of NOS, N-omega-methyl-l-arginine and asymmetric dimethylarginine, and resulted interestingly in a reduced ability of the protein inhibitor of nNOS (PIN) to dissociate nNOS dimers. We conclude that intracellular factors able to induce abnormalities in the nNOS monomer/dimer equilibrium could lead to pancreatic beta-cell dysfunction.
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PMID:Changes in the dimeric state of neuronal nitric oxide synthase affect the kinetics of secretagogue-induced insulin response. 1516 50

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.
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PMID:Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line. 1559 92

Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial gamma-carbonic anhydrases. A homozygous Arabidopsis knockout mutant carrying a T-DNA insertion in a gene encoding one of these subunits (At1g47260) was generated to investigate its physiological role. Isolation of mitochondria and separation of mitochondrial protein complexes by Blue-native polyacrylamide gel electrophoresis or sucrose gradient ultracentrifugation revealed drastically reduced complex I levels. Furthermore, the mitochondrial I + III2 supercomplex was very much reduced in mutant plants. Remaining complex I had normal molecular mass, suggesting substitution of the At1g47260 protein by one or several of the structurally related subunits of this respiratory protein complex. Immune-blotting experiments using polyclonal antibodies directed against the At1g47260 protein indicated its presence within complex I, the I + III2 supercomplex and smaller protein complexes, which possibly represent subcomplexes of complex I. Changes within the mitochondrial proteome of mutant cells were systematically monitored by fluorescence difference gel electrophoresis using 2D Blue-native/SDS and 2D isoelectric focussing/SDS polyacrylamide gel electrophoresis. Complex I subunits are largely absent within the mitochondrial proteome. Further mitochondrial proteins are reduced in mutant plants, like mitochondrial ferredoxin, others are increased, like formate dehydrogenase. Development of mutant plants was normal under standard growth conditions. However, a suspension cell culture generated from mutant plants exhibited clearly reduced growth rates and respiration. In summary, At1g47260 is important for complex I assembly in plant mitochondria and respiration. A role of At1g47260 in mitochondrial one-carbon metabolism is supported by micro-array analyses.
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PMID:Disruption of a nuclear gene encoding a mitochondrial gamma carbonic anhydrase reduces complex I and supercomplex I + III2 levels and alters mitochondrial physiology in Arabidopsis. 1593 78

In the mitochondrial permeability transition (MPT), MPT pores open to cause the mitochondrial inner membrane to become non-selectively permeable to molecules of mass up to 1500 Da. In this study, we used proteomics to investigate protein changes after MPT induction. Isolated rat liver mitochondria were incubated with various MPT inducers, including CaCl2, tert-butylhydroperoxide, and phenylarsine oxide, in the presence and absence of the MPT inhibitor, cyclosporin A. MPT induction was confirmed by an absorbance swelling assay. Mitochondrial membrane proteins prepared from control and treated mitochondria were separated by two-dimensional (2D) gel electrophoresis and stained with SyproRuby or Coomassie blue. Proteins of interest were further identified by mass spectrometry. 2D gel electrophoresis by isoelectric focusing and SDS-PAGE consistently showed a protein spot that shifted to a more basic isoelectric point after the MPT. This shift was prevented by CsA but did not occur after protonophoric uncoupling. Mass spectrometry identified this protein as the Rieske iron-sulfur protein (RISP) of ubiquinol-cytochrome c reductase (Complex III). Phosphatase treatment of sonicated mitochondria caused the same shift in RISP as occurred in MPT inducer-treated mitochondria. 2D gel electrophoresis by blue-native-PAGE and SDS-PAGE showed that RISP existed as an apparent monomer in mitochondrial membranes in addition to forming a complex with ubiquinol-cytochrome c reductase. These findings suggest that RISP may be part of MPT pores and that dephosphorylation of RISP may play a role in regulation of the MPT.
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PMID:Dephosphorylation of the Rieske iron-sulfur protein after induction of the mitochondrial permeability transition. 1602 95

The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.
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PMID:Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria. 1612 Apr 79

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a(2)b(2)) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n(H) = 0.55 +/- 0.05 for NADPH and 0.22 +/- 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.
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PMID:Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes. 1666 85

The role of insect saliva in the first contact between an insect and a plant is crucial during feeding. Some elicitors, particularly in insect regurgitants, have been identified as inducing plant defence reactions. Here, we focused on the salivary proteome of the green peach aphid, Myzus persicae. Proteins were either directly in-solution digested or were separated by 2D SDS-PAGE before trypsin digestion. Resulting peptides were then identified by mass spectrometry coupled with database investigations. A homemade database was constituted of expressed sequence tags from the pea aphid Acyrtosiphon pisum and M. persicae. The databases were used to identify proteins related to M. persicae with a nonsequenced genome. This procedure enabled us to discover glucose oxidase, glucose dehydrogenase, NADH dehydrogenase, alpha-glucosidase and alpha-amylase in M. persicae saliva. The presence of these enzymes is discussed in terms of plant-aphid interactions.
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PMID:Identification of aphid salivary proteins: a proteomic investigation of Myzus persicae. 1835 5

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.
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PMID:L-galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I. 1879 60

Membrane fraction of Methylococcus capsulatus (strain M) were treated with [14C]acetylene, an affinity label binding to the active center of membrane-bound methane monooxygenase (MMO). High-purity particulate form of methane hydroxylase (pMH) was obtained by ion exchange and hydrophobic chromatography. According to SDS-PAGE data, the enzyme contained three polypeptides with molecular weights of 47 (alpha), 27 (beta), and 25 (gamma) kDa in the ratio 1:1:1. The radiolabel was contained in the beta-subunit of pMH. The protein contained 1 or 2 atoms of nonheme iron and 2-4 atoms of copper per a minimum molecular weight of 99 kDa. This protein did not oxidize methane or propylene in the presence of NADH but was able to oxidize low quantities of methane in the presence of duroquinol. It was established that methanol dehydrogenase (MD) and NADH oxidoreductase (NADH-OR) are peripheral membrane proteins. Possible causes of low activity of high-purity methane hydroxylase are discussed.
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PMID:[Purification and properties of membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)]. 1894 92


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