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 gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function. This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain.
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PMID:Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase. 1167 11

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.
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PMID:Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study. 1197 22

NADPH-cytochrome P450 reductase, an obligatory component of the cytochrome P450 dependent monooxygenase system, was purified to electrophoretic homogeneity from beef liver microsomes. The purification procedure involved the ion exchange chromatography of the detergent-solubilized microsomes on first and second DEAE-cellulose columns, followed by 2',5'-ADP Sepharose affinity chromatography. Further concentration of the enzyme and removal of Emulgen 913 and 2'-AMP were accomplished on the final hydroxylapatite column. The enzyme was purified 239-fold and the yield was 13.5%. Monomer molecular weight of the enzyme was estimated to be 76000 +/- 3000 (N = 5) by SDS-PAGE. The absolute absorption spectrum of beef reductase showed two peaks at 455 and 378 nm, with a shoulder at 478 nm, characteristics of flavoproteins. The effects of cytochrome c concentration, pH, and ionic strength on enzyme activity were studied. Reduction of cytochrome c with the enzyme followed Michaelis-Menten kinetics, and the apparent K(m) of the purified enzyme was found to be 47.7 microM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer (pH 7.7). Stability of cytochrome c reductase activity was examined at 25 and 37 degrees C in the presence and absence of 20% glycerol. The presence of glycerol enhanced the stability of cytochrome c reductase activity at both temperatures. Sheep lung microsomal cytochrome P4502B and NADPH-cytochrome P450 reductase were also purified by the already existing methods developed in our laboratory. Both beef liver and sheep lung reductases were found to be effective in supporting benzphetamine and cocaine N-demethylation reactions in the reconstituted systems containing purified sheep lung cytochrome P4502B and synthetic lipid, phosphatidylcholine dilauroyl.
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PMID:Biochemical characteristics of purified beef liver NADPH-cytochrome P450 reductase. 1248 4

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

Pure mitochondria of the photosynthetic alga Chlamydomonas reinhardtii were analyzed using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The major oxidative phosphorylation complexes were resolved: F(1)F(0)-ATP synthase, NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase. The oligomeric states of these complexes were determined. The F(1)F(0)-ATP synthase runs exclusively as a dimer, in contrast to the C. reinhardtii chloroplast enzyme, which is present as a monomer and subcomplexes. The sequence of a 60-kD protein, associated with the mitochondrial ATP synthase and with no known counterpart in any other organism, is reported. This protein may be related to the strong dimeric character of the algal F(1)F(0)-ATP synthase. The oxidative phosphorylation complexes resolved by BN-PAGE were separated into their subunits by second dimension sodium dodecyl sulfate-PAGE. A number of polypeptides were identified mainly on the basis of their N-terminal sequence. Core I and II subunits of complex III were characterized, and their proteolytic activities were predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits in cytochrome c oxidase was demonstrated. Other mitochondrial proteins like the chaperone HSP60, the alternative oxidase, the aconitase, and the ADP/ATP carrier were identified. BN-PAGE was also used to approach the analysis of the major chloroplast protein complexes of C. reinhardtii.
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PMID:Identification of novel mitochondrial protein components of Chlamydomonas reinhardtii. A proteomic approach. 1274 37

Enzymatic reduction of physiological Fe(III) complexes of the "labile iron pool" has not been studied so far. By use of spectrophotometric assays based on the oxidation of NAD(P)H and formation of [Fe(II) (1,10-phenanthroline)3]2+ as well as by utilizing electron paramagnetic resonance spectrometry, it was demonstrated that the NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase (diaphorase, EC 1.8.1.4) effectively catalyzes the one-electron reduction of Fe(III) complexes of citrate, ATP, and ADP at the expense of the co-enzymes NAD(P)H. Deactivated or inhibited lipoyl dehydrogenase did not reduce the Fe(III) complexes. Likewise, in the absence of NAD(P)H or in the presence of NAD(P)+, Fe(III) reduction could not be detected. The fact that reduction also occurred in the absence of molecular oxygen as well as in the presence of superoxide dismutase proved that the Fe(III) reduction was directly linked to the enzymatic activity of lipoyl dehydrogenase and not mediated by O2. Kinetic studies revealed different affinities of lipoyl dehydrogenase for the reduction of the low molecular weight Fe(III) complexes in the relative order Fe(III)-citrate > Fe(III)-ATP > Fe(III)-ADP (half-maximal velocities at 346-485 microm). These Fe(III) complexes were enzymatically reduced also by other flavoenzymes, namely glutathione reductase (EC 1.6.4.2), cytochrome c reductase (EC 1.6.99.3), and cytochrome P450 reductase (EC 1.6.2.4) with somewhat lower efficacy. The present data suggest a (patho)physiological role for lipoyl dehydrogenase and other flavoenzymes in intracellular iron metabolism.
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PMID:Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool. 1296 36

NADH is central to the functioning of mitochondrial respiration. It is produced by enzymes in, or associated with, the tricarboxylic acid cycle in the matrix, and it is oxidized by two respiratory chain enzymes in the inner membrane, the rotenone-sensitive complex I and the rotenone-insensitive internal NADH dehydrogenase (ND(in)). A simplified kinetic model for NADH turnover in the matrix of plant mitochondria is presented. Only the two main NADH-producing enzymes, NAD-malate dehydrogenase [EC 1.1.1.37] (MDH) and NAD-malic enzyme [EC 1.1.1.39] (ME), are considered. This model reproduces the complex behaviour of malate oxidation by isolated mitochondria in response to additions of ADP (state 3/state 4), NAD(+) and/or rotenone, as well as to changes in pH. It is found that MDH always operates at or close to equilibrium. Changes in the activity of complex I, ND(in), or ME are predicted to cause clear changes in the pattern of malate oxidation. In general, the model predicts high sensitivity to changes in the ME activity. In contrast, MDH activity can be reduced 100-fold without detectable changes in malate oxidation. It is demonstrated that it is not the high activity, but the equilibrium properties of MDH that are important for the redox-buffering function of MDH in the mitochondrial matrix. Binding of NAD(+) and NADH in the matrix reduces the concentrations of free NAD(+) and NADH, depending on the concentration of binding sites and the binding strength. On the basis of the modelling results it is estimated that a significant proportion of the mitochondrial NAD is bound.
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PMID:Modelling NADH turnover in plant mitochondria. 1503 34

We report that oxidative phosphorylation and Ca2+ uptake processes are enhanced in liver mitochondria isolated from benzo[a]pyrene (B[a]P)-treated rats. The carcinogen did not affect either the respiratory control index or the Ca2+ control ratio. B[a]P treatment increased the oxidation rate of several substrates that donate electrons at the level of all three coupling sites, either the ADP- or Ca2+-stimulated rates or those observed after ADP or Ca2+ exhaustion. However, the efficiency of energy coupling was maintained because both ADP/O and Ca2+/site ratios remained unchanged. The electron flow through NADH-oxidase, NADH-duroquinone reductase, NADH-juglone reductase, NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome c oxidase was enhanced by B[a]P; however, succinate dehydrogenase activity was not affected. All these effects depended on the time post B[a]P administration, with a greater increase close to 48 h after administration of the carcinogen. The contents of cytochromes b, c1, and a + a3 from liver mitochondria, especially those isolated 48 h after B[a]P, were also significantly increased, although cytochrome c levels was just lightly increased 24 h after B[a]P treatment. These results suggest that B[a]P treatment stimulates mitochondrial respiration by increasing the level of several components of the mitochondrial respiratory chain. This may reflect mitochondrial adaptation to the cellular energy requirements of cell division in the neoplastic transformation process.
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PMID:Alterations of rat liver mitochondrial oxidative phosphorylation and calcium uptake by benzo[a]pyrene. 1520 43

The aim of the present study was to identify and characterize proteins of a 30- to 36-kDa fraction of Leishmania infantum promastigote membranes previously shown to be an immunodominant antigen(s) in Mediterranean visceral leishmaniasis (MVL) and a consistent and reliable serological marker of this disease. By the first approach, Coomassie-stained protein bands (32- and 33-kDa fractions) that specifically reacted by immunoblotting with sera from MVL patients were excised from the gel and submitted to enzymatic digestion to generate peptides. Four peptides were sequenced, three of which were shown to be definitely associated with MVL-reactive antigens and ascribed to a mitochondrial integral ADP-ATP carrier protein from L. major, a putative NADH cytochrome b(5) reductase, and a putative mitochondrial carrier protein, respectively. The second approach combined two-dimensional gel electrophoresis of membrane antigens and mass spectrometry (liquid chromatography-mass spectrometry/mass spectrometry) by using a quadrupole time-of-flight analysis. Six immunoreactive spots that resolved within a molecular mass range of 30 to 36 kDa and a pH range of 6.7 to 7.4 corresponded to four Leishmania products. The sequences derived from two spots were ascribed to a beta subunit-like guanine nucleotide binding protein, known as the activated protein kinase C receptor homolog antigen LACK, and to a probable member of the aldehyde reductase family. One spot was identified as a probable ubiquinol-cytochrome c reductase (EC 1.10.2.2) Rieske iron-sulfur protein precursor. The remaining three spots were identified as truncated forms of elongation factor 1alpha. These antigens correspond to conserved proteins ubiquitously expressed in eukaryotic cells and represent potential candidates for the design of a reliable tool for the diagnosis of this disease.
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PMID:Proteomic approach for characterization of immunodominant membrane-associated 30- to 36-kiloDalton fraction antigens of Leishmania infantum promastigotes, reacting with sera from Mediterranean visceral leishmaniasis patients. 1569 27

Chronic exposure to cigarette smoke affects the structure and function of mitochondria, which may account for the pathogenesis of smoking-related diseases. Bacopa monniera Linn., used in traditional Indian medicine for various neurological disorders, was shown to possess mitrochondrial membrane-stabilizing properties in the rat brain during exposure to morphine. We investigated the protective effect of bacoside A, the active principle of Bacopa monniera, against mitochondrial dysfunction in rat brain induced by cigarette smoke. Male Wistar albino rats were exposed to cigarette smoke and administered bacoside A for a period of 12 weeks. The mitochondrial damage in the brain was assessed by examining the levels of lipid peroxides, cholesterol, phospholipid, cholesterol/phospholipid (C/P) ratio, and the activities of isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase, and cytochrome C oxidase. The oxidative phosphorylation (rate of succinate oxidation, respiratory control ratio and ADP/O ratio, and the levels of ATP) was evaluated for the assessment of mitochondrial functional capacity. We found significantly elevated levels of lipid peroxides, cholesterol, and C/P ratio, and decreased levels of phospholipids and mitochondrial enzymes in the rats exposed to cigarette smoke. Measurement of oxidative phosphorylation revealed a marked depletion in all the variables studied. Administration of bacoside A prevented the structural and functional impairment of mitochondria upon exposure to cigarette smoke. From the results, we suggest that chronic cigarette smoke exposure induces damage to the mitochondria and that bacoside A protects the brain from this damage by maintaining the structural and functional integrity of the mitochondrial membrane.
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PMID:Protective effect of bacoside A on cigarette smoking-induced brain mitochondrial dysfunction in rats. 1605 Aug 6


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