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)

A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.
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PMID:Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning. 36 90

1. Incubation of NADH-ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH-K(3)Fe(CN)(6) oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH-cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH-K(3)Fe(CN)(6) oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.
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PMID:Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase from bovine heart mitochondria. 41 83

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.
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PMID:Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. 131 87

The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.
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PMID:Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803. 146 44

The structural gene of the Paracoccus denitrificans NADH-ubiquinone oxidoreductase encoding a homologue of the 75-kDa subunit of bovine complex I (NQO3) has been located and sequenced. It is located approximately 1 kbp downstream of the gene coding for the NADH-binding subunit (NQO1) [Xu, X., Matsuno-Yagi, A., and Yagi, T. (1991) Biochemistry 30, 6422-6428] and is composed of 2019 base pairs and codes for 673 amino acid residues with a calculated molecular weight of 73,159. The M(r) 66,000 polypeptide of the isolated Paracoccus NADH dehydrogenase complex is assigned the NQO3 designation on the basis of N-terminal protein sequence analysis, amino acid analysis, and immuno-cross-reactivity. The encoded protein contains a putative tetranuclear iron-sulfur cluster (probably cluster N4) and possibly a binuclear iron-sulfur cluster. An unidentified reading frame (URF3) which is composed of 396 base pairs and possibly codes for 132 amino acid residues was found between the NQO1 and NQO3 genes. When partial DNA sequencing of the regions downstream of the NQO3 gene was performed, sequences homologous to the mitochondrial ND-1, ND-5, and ND-2 gene products of bovine complex I were found, suggesting that the gene cluster carrying the Paracoccus NADH dehydrogenase complex contains not only structural genes encoding water-soluble subunits but also structural genes encoding hydrophobic subunits.
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PMID:Structural features of the 66-kDa subunit of the energy-transducing NADH-ubiquinone oxidoreductase (NDH-1) of Paracoccus denitrificans. 160 43

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. Structural genes encoding the subunits of this enzyme complex constitute at least one gene cluster [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428]. The 25-kDa subunit (NQO2), which has been isolated from sodium dodecyl sulfate-polyacrylamide gels, is a polypeptide of this enzyme complex. The partial N-terminal amino acid sequence and amino acid composition of the NQO2 subunit have been determined. On the basis of the amino acid sequence, the NQO2 gene was found to be located 1.7 kilobase pairs upstream of the gene for NADH-binding subunit (NQO1). The complete nucleotide sequence of the NQO2 gene was determined. It is composed of 717 base pairs and codes for 239 amino acid residues with a calculated molecular weight of 26,122. The NQO2 subunit is homologous to the Mr 24,000 subunit of the mammalian mitochondrial NADH-ubiquinone oxidoreductase which bears an electron paramagnetic resonance-visible binuclear iron-sulfur cluster (probably cluster N1b). Comparison of the predicted amino acid sequence of the Paracoccus NQO2 subunit with those of its mammalian counterparts suggests putative binding sites for the iron-sulfur cluster. In addition, nucleotide sequencing shows the presence of two unidentified reading frames between the NQO1 and NQO2 genes. These are designated URF1 and URF2 and are composed of 261 and 642 base pairs, respectively. The possible function of the protein coded for the URF2 is discussed.
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PMID:Characterization of the 25-kilodalton subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: sequence similarity to the 24-kilodalton subunit of the flavoprotein fraction of mammalian complex I. 190 71

A patient with chronic progressive external ophthalmoplegia (CPEO) who had abundant cytoplasmic bodies in muscle fibers and a deletion of mitochondrial DNA is reported. The patient was a 26-year-old male suffering from ophthalmoplegia from age 21. He had a marfanoid skeletal abnormality and perceptive hearing loss, but had neither retinopathy, ataxia, nor dementia. In the mitochondria isolated from the biopsied skeletal muscle, NADH-ubiquinone oxidoreductase activity was slightly decreased, succinate-cytochrome c reductase activity was slightly increased, and cytochrome c oxidase activity remained normal. Southern blot analysis of the muscle DNA identified heteroplasmy composed of a normal-sized mitochondrial DNA and a mutant mitochondrial DNA with a 4.2-kilobase deletion. The PCR plus S1 analysis showed that the deletion extended from nucleotide position 7860 +/- 60 to 12,090 +/- 70. The histological studies of the biopsied muscle revealed ragged-red fibers and cytochrome c oxidase-negative fibers in 15.7% and 18.6% of the muscle fibers, respectively. Other conspicuous histological change was abundant cytoplasmic bodies surrounded by clusters of abnormal mitochondria. The cytoplasmic bodies were found preferentially in type 1 fibers, and exclusively in cytochrome c oxidase-negative fibers and in ragged-red fibers. Focal existence of cytoplasmic bodies in muscle fibers with abnormal mitochondria suggests that segregated distribution of the abnormal mitochondria with deleted mitochondrial DNA is involved in the pathogenesis of cytoplasmic bodies.
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PMID:Cytoplasmic body and mitochondrial DNA deletion. 196 59

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. When the Paracoccus NADH dehydrogenase complex was irradiated by UV light in the presence of [adenylate-32P]NAD, radioactivity was incorporated exclusively into one of three polypeptides of Mr approximately 50,000. Similar results were obtained when [adenylate-32P]NADH was used. The labeling of the Mr 50,000 polypeptide was diminished when UV irradiation of the enzyme with [adenylate-32P]NAD was performed in the presence of NADH, but not in the presence of NADP(H). The labeled polypeptide was isolated by preparative sodium dodecyl sulfate gel electrophoresis and was shown to cross-react with antiserum to the NADH-binding subunit (Mr = 51,000) of bovine NADH-ubiquinone oxidoreductase. Its amino acid composition was also very similar to that of the bovine NADH-binding subunit. These chemical and immunological results indicate that the Mr 50,000 polypeptide is an NADH-binding subunit of the Paracoccus NADH dehydrogenase complex.
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PMID:Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans. 211 69

To investigate the molecular abnormality in the mitochondria from various tissues of an autopsied patient exhibiting mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, we have examined the enzymatic activity, iron-sulfur cluster, and subunit composition of the NADH-ubiquinone oxidoreductase (complex I). Rotenone-sensitive NADH-cytochrome c reductase activity was found to be decreased in all the tissues examined. A detailed study of the liver mitochondria has shown that NADH-ubiquinone oxidoreductase activity was greatly diminished. Analysis of the electron paramagnetic resonance spectra of the liver submitochondrial particles revealed a disproportionate deficiency of iron-sulfur clusters in the complex I segment of the respiratory chain. Signals from the clusters N-2 and N-3 diminished more drastically than those from clusters N-1b and N-4. Immunoblotting analysis showed that the 75-kD, 51-kD, and several other subunits were markedly diminished among multiple subunit polypeptides of complex I. These findings suggest that the underlying bases for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes are defects, at least, in the complex I subunits containing a flavin and/or iron-sulfur cluster(s), which resulted in deficiencies of some iron-sulfur clusters.
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PMID:Disproportionate deficiency of iron-sulfur clusters and subunits of complex I in mitochondrial encephalomyopathy. 249 47

From a high-salt extract of the purified thylakoid membrane, an 18-kD protein was detected. This protein was translated by the chloroplast ribosomes and could form a stable DNA-protein complex with a cloned chloroplast DNA replicative origin [Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) Mol. Gen. Genet. 209, 265-269]. In this paper, the 18-kD protein is linked to frxB, a chloroplast-encoded, ferredoxin-type, iron-sulfur protein, by N-terminal microsequencing of the purified protein and computer analysis. The identification is further supported empirically by the fact that the electron paramagnetic resonance spectra of the protein indicate the presence of iron-sulfur clusters. A polyclonal antibody raised against a synthetic pentadecameric peptide with amino acid sequence corresponds to the highly conserved region of the frxB protein and reacts strongly and specifically with the 18-kD protein band in protein gel blot analyses. The 18-kD iron-sulfur protein is found to be related to a subunit of the respiratory chain NADH dehydrogenase by its cross-reaction with a polyclonal antibody raised against highly purified NADH-ubiquinone oxidoreductase, a key enzyme of the respiratory chain. These data are consistent with chlororespiration, and, thus, possible implication of chlororespiration in regulating the initiation of chloroplast DNA replication is discussed.
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PMID:The 18-kD protein that binds to the chloroplast DNA replicative origin is an iron-sulfur protein related to a subunit of NADH dehydrogenase. 256 13


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