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At least 703 records · Page 39Linked to original sources
PubMed1990

Enzymatic synthesis of polyaniline film using a copper-containing oxidoreductase: bilirubin oxidase.

Electroactive polyaniline films have been synthesized by using a copper-containing oxidoreductase, bilirubin oxidase (BOD). Enzymatic polymerization took place on the surface of BOD-adsorbed solid matrix which was in contact with a buffer solution containing aniline. Optimum conditions for enzymatic polymerization of aniline were investigated. Elemental analysis and IR spectroscopy indicated that the enzymatically synthesized film was polyaniline. The cyclic voltammetric studies demonstrated that the polyaniline film was electrochemically reversible in the redox properties in acidic aqueous solutions. Since the film retained enzymatic activity of BOD which was employed as a catalyst for polymerization, enzymatic polymerization seems promising in preparation of immobilized enzyme membranes.

Aniline Compounds↗
PubMed1993

Phototransformation of monovinyl and divinyl protochlorophyllide by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

The chlorophyll precursors monovinyl protochlorophyllide (MV-PChlide) and divinyl protochlorophyllide (DV-PChlide) were extracted from mutant C-2A' of the unicellular green alga Scenedesmus obliquus which accumulates both protochlorophyllide derivatives in the dark. The two pigments were characterized by absorption and fluorescence spectroscopy and by plasma desorption mass spectrometry. The molecular masses of MV-PChlide and DV-PChlide were determined as 612 and 610 atomic mass units (amu) respectively. Both MV-PChlide and DV-PChlide were accepted as substrates and photoconverted to chlorophyllides in vitro by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

Chlorophyta↗
PubMed2005

A possible restriction of ferro- and ferricyanide oxidoreductase activities of rat liver mitochondria by the outer membrane.

In this work, various ferro-ferricyanide oxidoreductase activities of rat liver mitochondria were studied to find conditions under which the outer membrane might restrict the flux of these highly charged non-biological anions. When the isotonic low ionic strength medium was supplemented with 25mM KCl, a several-fold increase in the succinate-ferricyanide reductase activity of mitochondria and in the rate of external NADH oxidation in the presence of ferrocyanide was observed. Mitochondrial respiration with 5mM ferrocyanide was almost completely inhibited after consumption of 3.8-18.5% of the dissolved oxygen, depending on the medium and the presence of 2,4-dinitrophenol. These and other experimental data together with mathematical modeling of the redox-state equilibrium suggest that the measured activities might be restricted by two factors: first, the permeability of the outer mitochondrial membrane and second, a strong influence of the ionic strength of incubation media on the intermembrane space redox reactions.

Animals↗
PubMed2006

WW domain-containing oxidoreductase: a candidate tumor suppressor.

Common fragile site gene WWOX encodes a candidate tumor suppressor WW domain-containing oxidoreductase. Alteration of this gene, along with dramatic downregulation of WWOX protein, is shown in the majority of invasive cancer cells. Ectopic WWOX exhibits proapoptotic and tumor inhibitory functions in vitro and in vivo, probably interacting with growth regulatory proteins p53, p73 and others. Hyaluronidases regulate WWOX expression, increase cancer invasiveness and seem to be involved in the development of hormone-independent growth of invasive cancer cells. Estrogen and androgen stimulate phosphorylation and nuclear translocation of WWOX, although binding of WWOX to these sex hormones is unknown. We propose that suppression of WWOX expression by overexpressed hyaluronidases might contribute in part to the development of hormone independence in invasive cancer.

Biomarkers↗
PubMed2004

Expression of WW domain-containing oxidoreductase WOX1 in the developing murine nervous system.

WW domain-containing oxidoreductase WOX1, also known as WWOX or FOR, is a proapoptotic protein and a putative tumor suppressor. Hyaluronidases such as PH-20, Hyal-1 and Hyal-2 induce the expression of WOX1, and hyaluronidases and hyaluronan are involved in the embryonic development. In the present study, we document the expression of WOX1 in the developing murine nervous system. Immunohistochemical analysis revealed that WOX1 was differentially expressed in early dividing cells from all three germ layers from embryonic to perinatal stages. In murine fetuses, WOX1 was present prevalently in the brainstem, spinal cord and peripheral nerve bundles, but its expression decreased after birth. In parallel, the expression of WOX1, as determined by Western blotting, was significantly reduced in the brain stem and spinal cord of adult mice. Notably, high levels of WOX1 immunoreactivity was observed in the neural crest-derived structures such as cranial and spinal ganglia and cranial mesenchyme during the late fetal stage. In the adult brain, WOX1 is abundant in the epithelial cells of the choroids plexus and ependymal cells, while a low to moderate level of WOX1 is observed within white matter tracts, such as axonal profiles of the corpus callosum, striatum, optic tract, and cerebral peduncle. WOX1 is shown to mediate apoptosis synergistically with p53 in vitro. Nonetheless, the expression profiles of WOX1 were found to be similar in both p53 wild type and knockout mice, suggesting that WOX1 expression is not controlled by p53-mediated gene transcription. Taken together, in this study we have shown the expression and distribution of WOX1 in developing and adult murine nervous system. The potential role of WOX1 in the neuronal differentiation is discussed.

Aging↗
PubMed2005

Light-induced retinal damage involves tyrosine 33 phosphorylation, mitochondrial and nuclear translocation of WW domain-containing oxidoreductase in vivo.

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, is a known proapoptotic protein and a candidate tumor suppressor. Stress stimuli activate WOX1 via tyrosine 33 (Tyr33) phosphorylation and translocation to the mitochondria and nuclei in vitro. Here, the potential role of WOX1 in light-induced retinal degeneration in vivo was investigated. WOX1 is expressed primarily in the inner retina at perinatal stages, whereas an enhanced expression of WOX1, along with its Tyr33 phosphorylation (p-WOX1), is shown specifically in the retinal ganglion cells in adults. Prolonged exposure of mature rats to constant, low-intensity light (500 lux) for 1-2 months resulted in substantial death of photoreceptors and the presence of activated microglia, astrocytes and Muller glial in the outer retina. However, the inner retina was not or barely affected. In the damaged inner and outer nuclear layers of rat retina, WOX1 and p-WOX1 were overly expressed. Also, WOX1 colocalized with fragments of opsin-positive cones. In rd mice with an inherited retinal deficiency, upregulation of WOX1 and p-WOX1 in degenerated retina was observed with age. By electron microscopy, a large number of immunogold particles of WOX1 and p-WOX1 were found in the damaged mitochondria and condensed nuclei of degenerating photoreceptors, indicating that WOX1 undergoes activation and translocation to these organelles. In contrast, little or no WOX1-positive particles were found in the Golgi apparatus. In conclusion, activated WOX1 is likely to exert apoptosis of neuronal cells in the outer retina during the light-induced injury and in mice with an inherited retinal defect.

Active Transport, Cell Nucleus↗
PubMed2003

Quantitative amino acid analysis of bovine NADH:ubiquinone oxidoreductase (Complex I) and related enzymes. Consequences for the number of prosthetic groups.

Bovine-heart NADH:ubiquinone oxidoreductase (EC 1.6.5.3; Complex I) is the first and most complicated enzyme in the mitochondrial respiratory chain. Biochemistry textbooks and virtually all literature on this enzyme state that it contains one FMN and at least four iron-sulfur clusters. We show here that this statement is incorrect as it is based on erroneous protein determinations. Quantitative amino acid analysis of the bovine Complex I, to our knowledge the first reported thus far, shows that the routine protein-determination methods used for the bovine Complex I overestimate its protein content by up to twofold. The FMN content of the preparations was determined to be at least 1.3-1.4 mol FMN/mol Complex I. The spin concentration of the electron paramagnetic resonance (EPR) signal ascribed to iron-sulfur cluster N2 was determined and accounted for 1.3-1.6 clusters per molecule of Complex I. These results experimentally confirm the hypothesis [FEBS Lett. 485 (2000) 1] that the bovine Complex I contains two FMN groups and two clusters N2. Also the protein content of preparations of the soluble NAD(+)-reducing [NiFe]-hydrogenase (EC 1.12.1.2) from Ralstonia eutropha, which shows clear evolutionary relationships with Complex I, scores too high by the colorimetric protein-determination methods. Determination of the FMN content and the spin concentration of the EPR signal of the [2Fe-2S] cluster shows that this hydrogenase also contains two FMN groups. A third enzyme (Ech), the membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri which shows an even stronger evolutionary relationship with Complex I, behaves rather normal in protein determinations and contains no detectable acid-extractable FMN in purified preparations.

Amino Acids↗
PubMed2003

Chlorophylls of the c family: absolute configuration and inhibition of NADPH:protochlorophyllide oxidoreductase.

Using circular dichroism (CD) spectroscopy, the stereochemistry at C-13(2) of members of the chlorophyll (Chl) c family, namely Chls c(1), c(2), c(3) and [8-vinyl]-protochlorophyllide a (Pchlide a) was determined. By comparison with spectra of known enantiomers, all Chl c members turned out to have the (R) configuration, which is in agreement with considerations drawn from chlorophyll biosynthesis. Except for a double bond in the side chain at C-17, the chemical structure of Chl c(1) is identical with Pchlide a, the natural substrate of the light-dependent NADPH:protochlorophyllide oxidoreductase (POR). Thus, lack of binding to the active site due to the wrong configuration at C-13(2), which had been proposed previously, cannot be an explanation for inactivity of Chl c in this enzymic reaction. Our results show rather that Chl c(1) is a competitive inhibitor for this enzyme, tested with Pchlide a and Zn-protopheophorbide a (Zn-Ppheide a) as substrates.

Chlorophyll↗
PubMed1999

Enhanced neuronal expression of the oxidoreductase--biliverdin reductase--after permanent focal cerebral ischemia.

This is the first report on increased neuronal levels of biliverdin reductase (BVR) in response to ischemic brain injury. BVR is an oxidoreductase, and is unique among all enzymes characterized to date in having dual pH/dual cofactor requirements--NADH and NADPH at 6.7 and 8.7, respectively. BVR catalyses the final step in the heme metabolic pathway and reduces the heme degradation product, biliverdin, to bilirubin. Bilirubin can be both a neurotoxicant and an antioxidant depending on its ratio to protein and concentration. Bilirubin also has immunomodulatory activity. Other biologically active heme degradation products are iron and CO. This study assessed time-dependent changes in the level of BVR, following permanent middle cerebral artery occlusion (MCAo). It also examined correlation of the change in BVR expression with display of indices of ischemic tissue injury. Under halothane anesthesia and normothermic conditions, 72 DNX inbred mice were subjected to MCAo. A time-dependent enlargement of an ischemic lesion over the course of 24 h was observed and measured 55 +/- 5 mm3 at 6 h, 63 +/- 6.7 mm3 at 12 h, and 73 +/- 5 mm3 at 24 h. Six hours after MCAo, increased immunoreactivity for BVR was noted in neurons in the peri-ischemic areas, intraischemic cortical layers 3 and 5, as well as in neurons in regions distant from the borders of vascular distribution of the MCA, such as those in substantia nigra, in the Purkinje layer of the cerebellum and in the central nucleus of inferior colliculus. Twenty-four hours after MCAo, immunoreactivity for BVR remained increased in the peri-ischemia areas. At all time points staining for BVR was decreased in the ischemic core. At the 24 h time point there was an increase in Fe staining in the perimeter of the lesion and an increase in Schiff's staining for lipid peroxidation at the rim of the lesion. In situ hybridization analysis demonstrated a time dependent increase in BVR mRNA labeling in neurons of the peri-ischemic area. In the ischemic hemisphere, when compared with the contralateral hemisphere, neither measurable decreases in BVR mRNA or total protein levels nor a decrease in NADH-dependent BVR activity at pH 6.7 were observed. As judged by Northern and Western blots and activity analysis, despite the apparent loss of BVR from the ischemic core, and its increase in the peri-ischemic region, when compared with the contralateral hemisphere, the overall capacity of the ischemic hemisphere to catalyze the reduction of biliverdin was unchanged throughout the experiment. Should, in the case of ischemia, the conditions favor the antioxidant activity of bilirubin, then we suggest that increase in BVR expression in ischemic penumbra may present a cellular defense mechanism against free radical-mediated neuronal damage. Furthermore, we interpret the apparent tightly regulated expression of BVR in the ischemic hemisphere as an important factor in protection against bilirubin neurotoxicity. Data suggest that pharmacological modulation of BVR expression is a possible new direction for protecting neurons against ischemic injury and oxidative stress.

Animals↗
PubMed2000

Identification and light-induced expression of a novel gene of NADPH-protochlorophyllide oxidoreductase isoform in Arabidopsis thaliana.

In Arabidopsis thaliana, we identified a novel gene of a NADPH-protochlorophyllide oxidoreductase (POR) isoform, which catalyzes the light-dependent protochlorophyllide a reduction in the chlorophyll (Chl) biosynthetic pathway. The deduced amino acid sequence of the novel POR isoform (PORC) showed significant identities ( approximately 75%) with the previously isolated two POR isoforms of A. thaliana. Contrasting with these POR isoforms, the PORC transcript increased in etiolated seedlings by illumination, and was dominantly expressed in immature and mature tissues. The present results demonstrated that Chl biosynthesis and chloroplast biogenesis in A. thaliana are controlled by three POR isoforms, which are differentially controlled by light and development.

Amino Acid Sequence↗
PubMed2000

The influence of glycerol and chloroplast lipids on the spectral shifts of pigments associated with NADPH: protochlorophyllide oxidoreductase from Avena sativa L.

Dark-grown angiosperm seedlings lack chlorophylls, but accumulate protochlorophyllide a complexed with the light-dependent enzyme NADPH:protochlorophyllide oxidoreductase. Previous investigators correlated spectral heterogeneity of in vivo protochlorophyllide forms and a shift of chlorophyllide forms from 680 to 672 nm (Shibata shift) occurring after irradiation, with intact membrane structures which are destroyed by solubilization. We demonstrate here that the various protochlorophyllide forms and the Shibata shift which disappear upon solubilization are restored if the reconstituted complex is treated with plastid lipids and 80% (w/v) glycerol. We hypothesize that the lipids can form a cubic phase and that this is the precondition in vitro and in vivo for the observed spectral properties before and after irradiation.

Avena↗
PubMed2000

NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation.

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexahistidine tag at the N-terminus. This enzyme (His(6)-POR) has been purified to homogeneity and a preliminary characterisation of its kinetic and substrate binding properties is presented. Chemical modification experiments have been used to demonstrate inhibition of POR activity by the thiol-specific reagent N-ethyl maleimide. Substrate protection experiments reveal that the modified Cys residues are involved in either substrate binding or catalysis.

Chlorophyllides↗
PubMed2001

The importance of the C-terminal region and Cys residues for the membrane association of the NADPH:protochlorophyllide oxidoreductase in pea.

In vitro chloroplast import reactions and thylakoid association reactions have been performed with a series of C-terminal deletions and Cys-to-Ser substitution mutants of the pea NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99). C-terminal deletions of the precursor POR (Delta362-400, Delta338-400, Delta315-400 and Delta300-400) were efficiently translocated across the chloroplast envelope. However, except the Delta396-400 mutant, no C-terminal deletion mutants or Cys-to-Ser substitution (Cys119, Cys281 and Cys309) mutants resisted post-treatment with thermolysin after the thylakoid association reactions. This suggests that these mutants were unable to properly associate to the thylakoids due to changes of the protein conformation of POR.

Binding Sites↗
PubMed1998

F420H2:NADP oxidoreductase from Methanobacterium thermoautotrophicum: identification of the encoding gene via functional overexpression in Escherichia coli.

F420H2:NADP oxidoreductase is found in methanogenic, sulfate-reducing and halophilic archaea and also in some bacteria. The putative gene encoding the enzyme was cloned from Methanobacterium thermoautotrophicum (strain Marburg) and heterologously expressed in Escherichia coli. The overproduced active enzyme was purified, characterized and crystallized.

Chromatography↗
PubMed1997

Primary structure of an invertebrate dihydrolipoamide dehydrogenase with phylogenetic relationship to vertebrate and bacterial disulfide oxidoreductases.

Dihydrolipoamide dehydrogenase (E3) is a flavoprotein component of multi-enzyme complexes catalyzing oxidative decarboxylation of alpha-ketoacids in the Krebs' cycle. We have cloned a 2.4-kb E3 cDNA from an arthropod, Manduca sexta, that codes for 497 amino acids and translates to a 51-kDa protein in vitro. Sequences at and around the dinucleotide binding domains, disulfide active site and the C-terminal interface domain involved in substrate binding are highly conserved in Manduca E3. Phylogenetic analysis of protein sequences from the flavoprotein class of disulfide oxidoreductases family of enzymes suggests that in spite of the homologous nature of E3 and glutathione reductase (goR) in sequence and structure, E3 shares a common ancestor with mercuric reductase (merA), whereas goR is more related to trypanothione reductase (tryR) than to other members. All members, except goRs, seemed to be monophyletic. Plant goRs seemed to have arisen differently and are more closely related to tryRs than to bacterial and vertebrate goRs. Earlier speculation on the nature of origin of E3 in Pseudomonas is not supported by phylogenetic data. A possible structural relationship of Manduca E3 to other pyridine-binding proteins, such as the neurotransmitter transporters and channels, is proposed.

Amino Acid Sequence↗
PubMed2001

Identification of two tetranuclear FeS clusters on the ferredoxin-type subunit of NADH:ubiquinone oxidoreductase (complex I).

The proton-translocating NADH:ubiquinone oxidoreductase of respiratory chains (complex I) contains one flavin mononucleotide and five EPR-detectable iron-sulfur clusters as redox groups. Because of the number of conserved motifs typical for binding iron-sulfur clusters and the high content of iron and acid-labile sulfide of complex I preparations, it is predicted that complex I contains additional clusters which have not yet been detected by EPR spectroscopy. To search for such clusters, we used a combination of UV/vis and EPR spectroscopy to study complex I from Neurospora crassa and Escherichia coli adjusted to distinct redox states. We detected a UV/vis redox difference spectrum characterized by negative absorbances at 325 and 425 nm that could not be assigned to the known redox groups. Redox titration was used to determine the pH-independent midpoint potential to be -270 mV, being associated with the transfer of two electrons. Comparison with UV/vis difference spectra obtained from complex I fragments and related enzymes showed that this group is localized on subunit Nuo21.3c of the N. crassa or NuoI of the E. coli complex I, respectively. This subunit (the bovine TYKY) belongs to a family of 8Fe-ferredoxins which contain two tetranuclear iron-sulfur clusters as redox groups. We detected EPR signals in a fragment of complex I which we attribute to the novel FeS clusters of complex I.

Animals↗
PubMed1976

Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance.

A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases. The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes. Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively. In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.

Alcohol Oxidoreductases↗
PubMed2001

Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state.

Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protochlorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.

Amino Acid Substitution↗