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)

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.
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PMID:Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase. 1205 81

The activity of the hepatic oxidative drug metabolizing system has been investigated in an experimentally-induced heat stress animal model pretreated with phenobarbitone. Female rats, unacclimatized and untrained were pretreated for 3 days with phenobarbitone as the inducing agent for the drug metabolizing systems. On the fourth day, they were restrained and exposed to an ambient temperature of 40 degrees C. One hour after acute exposures to such conditions, the activities of hepatic cytochrome P450, cytochrome b5 and NADPH cytochrome c reductase were significantly decreased in the induced animal model. Further, cytochrome P450 isozymes observed by SDS-gel electrophoresis were significantly decreased. In addition, the hypnotic effect of pentobarbitone was significantly increased. It is concluded that the activity of the hepatic oxidative drug metabolizing enzymes was decreased in induced drug metabolism systems exposed to heat stress conditions.
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PMID:The effect of heat stress on the induced hepatic drug metabolizing system in rats. 1206 73

Reservosomes are acidic compartments present at the posterior region of epimastigote forms of Trypanosoma cruzi that store proteins and lipids. During metacyclogenesis, they consume their contents and disappear. Reservosomes are rich in cruzipain, the main proteolytic enzyme of this parasite. By centrifugation in a sucrose gradient, we have obtained a highly purified subcellular fraction containing reservosomes from 5-day-old Y strain epimastigotes. Transmission electron microscopy showed that the fraction contained well-preserved organelles. The protein profile of the organelle analyzed by SDS-PAGE depicted a wide range of protein bands, predominating those corresponding to a triplet of 60-51 kDa and a doublet of 25-23 kDa. Protease activity in substrate-containing gels, in the presence or absence of protease inhibitors, showed that cysteine proteinase is enriched and very active in the purified fraction. Enzymatic assays demonstrated the absence of pyrophosphatase, an acidocalcisome marker, and succinate cytochrome c reductase, a mitochondrial marker, although these enzymes were active in other regions of the purification sucrose gradient. Thin layer chromatographic neutral lipid analysis of purified reservosomes demonstrated that the organelle stores large amounts of ergosterol and esterified cholesterol. Phospholipid analysis indicated phosphatidylcholine and phosphatidylethanolamine as the major constituents of reservosome membranes.
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PMID:Isolation and characterization of a reservosome fraction from Trypanosoma cruzi. 1220 65

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

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.
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PMID:Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase. 1279 75

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


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