Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ICRF-187 (dexrazoxane) is currently in clinical trials as a cardioprotective agent for the prevention of doxorubicin-induced cardiotoxicity. ICRF-187 likely acts through its strongly metal ion-binding rings-opened hydrolysis product ADR-925 by removing iron from its complex with doxorubicin or by chelating free iron. The ability of NADPH-cytochrome-P450 reductase to promote hydroxyl radical formation by iron complexes of ADR-925 and EDTA was compared by EPR spin trapping. The iron-EDTA complex produced hydroxyl radicals at six times the rate that the iron-ADR-925 complex did. The aerobic oxidation of ferrous complexes of ADR-925, its tetraacid analog, EDTA and DTPA was followed spectrophotometrically. The iron(II)-ADR-925 complex was aerobically oxidized 700 times slower than was the EDTA complex. It is concluded that even though ADR-925 does not completely eliminate iron-based hydroxyl radical production, it likely protects by preventing site-specific hydroxyl radical damage by the iron-doxorubicin complex.
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PMID:NADPH-cytochrome-P450 reductase promotes hydroxyl radical production by the iron complex of ADR-925, the hydrolysis product of ICRF-187 (dexrazoxane). 763 62

Luteoskyrin is a hepatotoxic and hepatocarcinogenic bisdihydroanthraquinone produced by Penicillium islandicum Sopp. By observing the EPR spectra of DMPO-spin adducts and luteoskyrin semiquinone radical, we investigated in vitro whether luteoskyrin is reduced to its semiquinone radical leading to the generation of active oxygen species in redox systems catalyzed by NADPH-dependent cytochrome reductases of the liver. We found (1) the formation of luteoskyrin semiquinone radical in the NADPH-cytochrome P-450 reductase system under anaerobic conditions, (2) the generation of O2- in the systems composed of luteoskyrin, NAD(P)H, and either rat liver microsomal NADPH-cytochrome P-450 reductases or submitochondrial particles and (3) dicoumarol showed no effect on the O2- generation in the case of submitochondrial particles. From these results we proposed that luteoskyrin liver injuries are induced by the active oxygen species generated in the process of autoxidation of luteoskyrin semiquinone radical which is produced in the one-electron redox systems catalyzed by the liver NAD(P)H-dependent cytochrome reductases.
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PMID:Spin-trapping and direct EPR investigations on the hepatotoxic and hepatocarcinogenic actions of luteoskyrin, an anthraquinoid mycotoxin produced by Penicillium islandicum Sopp. Generations of superoxide anion and luteoskyrin semiquinone radical in the redox systems consisted of luteoskyrin and liver NADPH- or NADH-dependent reductases. 764 18

Adriamycin (Adr) is one of the most powerful antitumor drugs. Its therapeutic effect may be due to its cyclic reduction-oxidation and, thus, generation of oxygen radicals. Using the spin-trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and EPR we have demonstrated that in an enzymatic system consisting of NADPH, NADPH-cytochrome P-450 reductase, and Fe(EDTA)2 Adr stimulates formation of .OH radicals in the presence of DNA or RNA with equal efficiency. Incubation of nucleic acids in the Adr-dependent reaction generating .OH radicals resulted in extensive degradation of double- and single-stranded DNA, but did not effect RNA. In contrast, both DNA and RNA were effectively destroyed in a footprinting system, ascorbate-Fe(EDTA)2-H2O2, which generates .OH radicals in massive quantities. Fluorescence assays indicated that Adr forms stable complexes with ds- and ss-DNA but reacts only slightly with RNA. We conclude that the formation of Adr-nucleic acid complex is necessary for .OH radical-mediated cleavage of the latter, and thus, Adr may be regarded as a chemical nuclease acting in situ.
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PMID:Dependence of nucleic acid degradation on in situ free-radical production by adriamycin. 769 53

The well-known metabolism of CCl4 to trichloromethyl radicals in rat liver microsomal dispersions has been reinvestigated with the goal to determine the repeatability and reproducibility of the EPR signal intensity of the EPR spectrum of the CCl3 adduct of PBN. It was found that at least eight repeat experiments were needed under identical conditions to obtain an average value with an error of +/- 10%. When the effect of changing the concentrations of CCl4, PBN or NADPH-generating system was investigated, the plots of EPR signal intensity vs. the variable selected showed initial smooth increases in signal strength with respect to an increase in concentrations of CCl4, PBN or NADPH-generating system. However, considerable scatter was found after the initial slope and only general trends could be recognized. It is concluded that with CCl4, no increase in EPR signal is found after 10 mM concentration. For PBN, the optimum concentration is about 30 mM. The signal strength seems to increase with increased amounts of NADPH generating system although with diminishing slope.
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PMID:Study of reproducibility of spin trapping results in the use of C-phenyl-N-tert-butyl nitrone (PBN) for trichloromethyl radical detection in CCl4 metabolism by rat liver microsomal dispersions. Biological spin trapping I. 769 99

A truncated, soluble rat heme oxygenase-1 lacking its C-terminal, membrane-anchoring segment, and its His25-->Ala and His132-->Ala mutants have been prepared by site-directed mutagenesis and expression in Escherichia coli. We found that wild-type enzyme can degrade heme to biliverdin, but its specific activity was about one-fifth that of the native, full-length enzyme, suggesting that the C-terminal segment is important for accepting electrons from NADPH cytochrome P450 reductase. His132-->Ala mutant had an enzyme activity comparable to that of the wild-type enzyme; hence, the highly conserved His132 is not essential for the display of the heme oxygenase activity. In contrast, His25-->Ala mutation completely abolished the enzyme's catalytic activity. A five-coordinate type ferrous NO EPR spectrum was observed for the heme-heme oxygenase H25A complex. Hence, we conclude that His25 is the proximal axial ligand of the heme iron and is essential for the heme degradation activity of the enzyme.
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PMID:Demonstration that histidine 25, but not 132, is the axial heme ligand in rat heme oxygenase-1. 787 92

The generation of hydroxyl radicals by rat liver microsomes was monitored by spin trapping with 5,5-dimethylpyrroline N-oxide (DMPO). The results confirm and extend previous data which demonstrated that hydroxyl radicals are produced by microsomes in the presence of NADPH and O2, and without the exogenous addition of iron. No EPR signals could be detected unless catalase activity which was associated with the microsomes could be substantially diminished. Addition of azide was the most effective means of eliminating catalase activity, but azide also reacted rapidly with hydroxyl radicals, forming azidyl radicals which were in turn trapped by DMPO. Extensive washing and preincubation of microsomes with 3-amino-1,2,4-triazole in the presence of H2O2 were evaluated as alternative methods of decreasing the catalase contamination of microsomes. Although neither method completely eliminated microsomal catalase activity, addition of azide was no longer necessary for hydroxyl radical detection with DMPO. When highly washed microsomal preparations were tested, weak signals of the superoxide radical adduct of DMPO could also be detected. These data indicate that the sensitivity of spin trapping in microsomal systems can be improved substantially when care is taken to eliminate cytosolic contaminants such as catalase.
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PMID:Oxygen radical formation in well-washed rat liver microsomes: spin trapping studies. 801 21

Toxicosis due to paraquat, a redox cycling xenobiotic, is still a subject of much debate. In the present study on lipid peroxidation, paraquat had a biphasic effect on the malondialdehyde (MDA) level in rat liver microsomes; stimulation at the initial stage (within 10 min) and depression at the later stage. Although paraquat increased the initial rate of NADPH oxidation dose-dependently, the rate was not necessarily parallel with the increase in the MDA level. The MDA level increased linearly up to 0.1 mM paraquat added, but then it attained a plateau. The stimulation obtained by paraquat within 10 min was absolutely dependent on exogenous Fe2+ ion and NADPH, and the stimulation was entirely SOD sensitive, while the iron-driven increase in MDA was 20% sensitive. Thus, there were different mechanisms between iron-driven lipid peroxidation and paraquat-modified peroxidation. Catalase increased the level, but mannitol, a scavenger of OH, had no effect. EPR spectra showed that superoxide was formed dose-dependently up to 0.1 mM paraquat and that it attained a plateau at the same as MDA level described above. From these results, we concluded that paraquat stimulates lipid peroxidation through a mechanism dependent on the superoxide complex involving Fe2+ ion.
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PMID:Effect of paraquat on the malondialdehyde level in rat liver microsomes (in vitro). 802 66

Molecular oxygen or cytochrome c has been described as the electron acceptor of the reaction of old yellow enzyme with NADPH. In this study, menadione was found to be a sensitive electron acceptor of the reaction under aerobic as well as anaerobic conditions. The Km value of menadione for old yellow enzyme is as low as 2-3 x 10(-7) M in the presence or absence of superoxide dismutase. The rate enhancement of the cytochrome c reduction of old yellow enzyme with NADPH was about eight times in the presence of menadione. The rate increment was slightly higher under aerobic than anaerobic conditions. The rate enhancement by menadione enabled sensitive determination of the enzyme activity in the assay system, which contained NADPH, cytochrome c, menadione, and old yellow enzyme. In the reaction course, the semiquinone species of menadione was trapped by the reaction with t-butyl-alpha-phenylnitrone. The radical adduct was detected on EPR. The dyestuff, 2,6-dichlorophenolindophenol, was found to be reduced ineffectively even in the presence of menadione; moreover, it was inhibitory in the NADPH consumption reaction. Methylene blue or Lauth's violet, known to be capable of semiquinone formation, also behaved, like menadione, as a mediator of electron transport to cytochrome c. On the basis of the experimental results, the occurrence of the one electron transfer of the old yellow enzyme reaction was emphasized.
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PMID:Characterization of the electron acceptors of old yellow enzyme: mechanistic approach to the mode of one electron transfer from the enzyme to menadione or dyestuffs. 813 46

We report here the first purification of the protein encoded by the Escherichia coli bioB gene. One species of this protein runs on native gels with an electrophoretic mobility typical of a protein with m = 82 kDa, suggesting the protein is a dimer (gene sequence predicts m = 38.7 kDa). There are two iron- and two acid-labile sulfur atoms per protein monomer. Solutions containing the protein are red and have an absorbance spectrum characteristic of proteins with [2Fe-2S] clusters. In its oxidized native state, the protein is EPR-silent. Upon addition of dithionite, the protein's UV-visible absorbance spectrum is very slowly bleached, and an EPR active species is produced that displays a signal at gavg = 1.95. All these results are consistent with this protein containing one [2Fe-2S] cluster per monomer. The EPR spin quantitation is only 5-10% of expected. Since this protein loses iron upon reduction with dithionite, the low-spin quantitation is probably due to cluster instability in the reduced state. Another species of the bioB gene products has also been purified which runs on native gels with an electrophoretic mobility typical of a protein with m = 104 kDa. This species appears to be a dimer with one [2Fe-2S] cluster per dimer. The 104-kDa protein can be converted to the 82-kDa protein upon incubation with Fe3+ and S2-. The bioB gene product we have isolated is active in the conversion of dethiobiotin to biotin in vitro in the presence of NADPH, AdoMet, Fe3+ or Fe2+, and additional unidentified factors from the crude extracts of E. coli. The Km for dethiobiotin in this reaction has been found to be 2 microM.
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PMID:Biotin synthase: purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product. 814 61

EPR spin trapping has been employed to directly detect radical production in isolated rat liver nuclei on exposure to a variety of hydroperoxides and related compounds which are known, or suspect, tumour promoters. The hydroperoxides, in the absence of reducing equivalents, undergo oxidative cleavage, generating peroxyl radicals. In the presence of NADPH (and to a lesser extent NADH) reductive cleavage of the O-O bond generates alkoxyl radicals. These radicals undergo subsequent rearrangements and reactions (dependent on the structure of the alkoxyl radical), generating carbon-centred radicals. Acyl peroxides and peracids appear to undergo only reductive cleavage of the O-O bond. With peracids this cleavage can generate aryl carboxyl (RCO2.) or hydroxyl radicals (HO.); with acyl peroxides, aryl carboxyl radicals are formed and, in the case of t-butyl peroxybenzoate, alkoxyl radicals (RO.). The radicals detected with each peroxide are similar in type to those detected in the rat liver microsomal fraction, although the extent of radical production is lower. The subsequent reactions of the initially generated radicals are similar to those determined in homogeneous chemical systems, suggesting that they are in free solution. Experiments with NADPH/NADH, heat denaturation of the nuclei and various inhibitors suggest that radical generation is an enzymatic process catalysed by haemoproteins, in particular cytochrome P-450, and that NADPH/cytochrome P-450 reductase is involved in the reductive cleavage of the O-O bond. The generation of these radicals by the rat liver nuclear fraction is potentially highly damaging for the cell due to the proximity of the generating source to DNA. Several previous studies have shown that some of the radicals detected in this study, such as aryl carboxyl and aryl radicals, can damage DNA, via various reactions which result in the generation of strand breaks and adducts to DNA bases: these processes are suggested to play an important role in the tumour promoting activity of these hydroperoxides and related compounds.
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PMID:Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds. 815 40


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