UNIPROT:Q8NEX9 - FACTA Search

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Both purified cytochrome P-450 (P-450) and free ferriprotoporphyrin IX are destroyed by NADPH-P-450 reductase in the presence of NADPH and O2. The process appears to be mediated by H2O2 generated by reduction of O2. Six major products were identified from the reaction of H2O2 with ferri-protoporphyrin IX-hematinic acid, methylvinylmaleimide, and four dipyrrolic propentdyopents. The structures of the propentdyopents were elucidated by mass spectrometry and 1H NMR methods. Both free ferriprotoporphyrin IX and P-450 yielded these same products in similar relative ratios. P-450 heme in rat liver microsomes was degraded in the presence of O2 and NADPH and either NaN3 (a catalase inhibitor) or Fe-ADP (which promotes lipid peroxidation); the products were primarily hematinic acid, methylvinylmaleimide, and small quantities of one propentdyopent. Only the two maleimides were detected in the destruction of microsomal P-450 heme by cumene hydroperoxide and iodosylbenzene. On the basis of the reaction of H2O2 with several metal-octaethylethylporphyrin complexes and free octaethylporphyrin, the iron chelated in ferriprotoporphyrin IX is required for degradation by H2O2. Biliverdin is not an intermediate in the formation of maleimides and propentdyopents from heme. Experiments using the tetraethylpropentdyopent produced from ferrioctaethylporphyrin suggest that propentdyopents are not further cleaved to form the maleimides. A mechanism for oxidative heme destruction consistent with these observations is proposed.
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PMID:Characterization of the enzymatic and nonenzymatic peroxidative degradation of iron porphyrins and cytochrome P-450 heme. 392 75

The antioxidative potential of cyanobacteria to scavenge hydroperoxides formed as by-products of photosynthetic activity was investigated in Nostoc muscorum 7119 and Synechococcus 6311. These cells contained a high concentration of glutathione, 2-5 mM, and a low concentration of ascorbate, 20-100 uM. No glutathione peroxidase was detected while the activity of ascorbate peroxidase was high, reacting with hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide. Dehydroascorbate reductase was active in recycling ascorbate and glutathione reductase regenerated glutathione from glutathione disulphide. The activity of these antioxidative enzymes in the cyanobacteria was sufficient to remove between 60-230 nmoles H2O2 .mg protein-1 min-1. It is suggested that in cyanobacteria an effective reaction sequence for removal of hydroperoxides involves ascorbate peroxidase and recycling of glutathione and ascorbate.
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PMID:The role of glutathione and ascorbate in hydroperoxide removal in cyanobacteria. 393 3

The FAD binding site of rabbit liver glutathione reductase has been explored by reconstitution of the apoprotein with several FAD analogs modified in the isoalloxazine ring. The apoglutathione reductase binds the p-quinoid form of 8-mercapto-FAD, suggesting that the protein stabilizes a negative charge in the -N1-C2 = O position of the pyrimidine subnucleus. The main absorption peak in the visible spectrum of the 8-mercapto-FAD-enzyme is at 585 nm; treatment of the reconstituted protein with reducing agents of disulfide groups induces a reversible hypochromic shift of 20 nm of the peak. Thus, in 8-mercapto-FAD-glutathione reductase, the oxidation-reduction state of the active center disulfide can be monitored. The chemical reactivity toward methylmethanethiosulfonate and iodoacetamide of the 8-mercapto-FAD-enzyme shows that the flavin position 8 is freely accessible to solvent. However, position 2 is buried within the protein molecule as judged from the lack of reactivity of the 2-thio-FAD-enzyme with methylmethanethiosulfonate. Hydrogen peroxide reacts slowly with both 2-thio-FAD-enzyme and native glutathione reductase, yielding inactive enzyme with a modified spectrum; the prosthetic group is still protein bound. Differences in the active site of the rabbit liver enzyme compared to the human erythrocyte glutathione reductase are evidenced by use of FAD analogs: the peaks of reconstituted liver enzymes are shifted about 10 nm toward longer wavelengths.
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PMID:Properties of rabbit liver glutathione reductase reconstituted with FAD analogs. 394 91

Reactions of human oxyhemoglobin A with iron(II) compounds have been investigated. Human oxyhemoglobin (HbO2) reacts with aquopentacyanoferrate(II), Fe(II)(CN)5H2O3-, to yield hydrogen peroxide, aquomethemoglobin and Fe(III)(CN)5H2O2-. The reaction follows a second order rate law, first order in the pentacyanide and in HbO2. Since reaction rates are lower in the presence of catalase, the H2O2 produced must promote metHb formation in reactions independent of pentacyanide. Changes in concentrations of effectors (e.g. H+, inositol hexaphosphate, Cl-, and Zn2+), alkylation of beta-93 cysteine with N-ethylmaleimide, and substitution at distal histidine (as in Hb Zurich with beta-63 His----Arg) in each case can markedly affect pentacyanide reaction rates demonstrating a fine control of rates by protein structure. Hexacyanoferrate(II) (ferrocyanide) reacts with HbO2 to produce cyano-metHb as well as aquo-metHb but the reaction with the hexacyanide is much slower than with the aquopentacyanide. Iron(II) EDTA converts HbO2 to deoxy-Hb with no evidence for formation of metHb as an intermediate. These findings support a mechanism in which the pentacyanide anion reacts directly with coordinated dioxygen. One-electron transfers to O2 from both pentacyanide iron(II) and heme iron(II) result in the formation of a mu-peroxo intermediate, HbFe(III)-O-O-Fe(III) (CN)5(3-). Hydrolysis of this intermediate yields metHb . H2O, H2O2, and FeIII(CN)5H2O2-. The reaction of HbO2 with Fe(CN)6(4-) must follow an outer sphere electron transfer mechanism. However, the very slow rate that is seen with Fe(CN)6(4-) could arise entirely from the pentacyanide produced from loss of one cyanide ligand from the hexacyanide. Fe(II)EDTA reacts rapidly with free O2 in solution but can not interact directly with the heme-bound O2 of HbAO2. The dynamic character of the O2 binding sites apparently permits access of the Fe2+ of the pentacyanide to coordinated dioxygen but the protein structure is not sufficiently flexible to allow the larger Fe2+EDTA molecule to react with bound O2. It is necessary for maintenance of the oxygen transport function of the red cell for reductants such as the methemoglobin reductase system, glutathione, and ascorbate to be able to reduce metHb to deoxy-Hb. It is also important for these reductants to be unable to donate an electron to HbO2 to yield H2O2 and metHb. Thus, a mechanistic requirement for the delivery of one-electron directly to the dioxygen ligand, if peroxide is to be produced, enables the protein to protect the oxygenated species from those electron donors normally present in the cell by denying these reductants steric access to coordinated O2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of electron transfer to coordinated dioxygen of oxyhemoglobins to yield peroxide and methemoglobin. Protein control of electron donation by aquopentacyanoferrate(II). 398 29

In thyroid gland, iodination takes place on the apical plasma membrane and requires the presence of the thyroid peroxidase and H2O2 generating system. H2O2 generation and NBT (nitro blue tetrazolium) reductase activity (both of which are NADPH-dependent) as well as peroxidase activity were compared for their respective orientations in membrane vesicles. The possible role of NADPH-NBT reductase activity in H2O2 generation was also examined. Results favor the conclusion that thyroid peroxidase is oriented towards the luminal side of the vesicles, whereas the NADPH site of NADPH oxidase-dependent H2O2 generation is located on the external side of the same or of different vesicles. Furthermore, it is shown that different NADPH-NBT reductase activities are present on both the outer and inner surfaces of the membrane vesicles, and that none of these activities is able to produce either H2O2 or O-2. The idea that a multi-component complex is involved in H2O2 generation is discussed, and a model is proposed which takes into account the possible spatial separation of the thyroid peroxidase site from the NADPH site of this H2O2 generation system on the apical membrane of the thyrocyte.
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PMID:Relation between thyroid peroxidase, H2O2 generating system and NADPH-dependent reductase activities in thyroid particulate fractions. 401 97

The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88-97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5-6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H2O2 generated in situ from a glucose oxidase system; in each case Vmax is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased Km, is lowered. The spin state of purified yeast cytochrome P-448 was 94% low spin (22 degrees C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spin at 22 degrees C). Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from beta-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase, from Saccharomyces cerevisiae. 632 93

Uninduced rat liver microsomes and NADPH-Cytochrome P-450 reductase, purified from phenobarbital-treated rats, catalyzed an NADPH-dependent oxidation of hydroxyl radical scavenging agents. This oxidation was not stimulated by the addition of ferric ammonium sulfate, ferric citrate, or ferric-adenine nucleotide (AMP, ADP, ATP) chelates. Striking stimulation was observed when ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (DTPA) was added. The iron-EDTA and iron-DTPA chelates, but not unchelated iron, iron-citrate or iron-nucleotide chelates, stimulated the oxidation of NADPH by the reductase in the absence as well as in the presence of phenobarbital-inducible cytochrome P-450. Thus, the iron chelates which promoted NADPH oxidation by the reductase were the only chelates which stimulated oxidation of hydroxyl radical scavengers by reductase and microsomes. The oxidation of aminopyrine, a typical drug substrate, was slightly stimulated by the addition of iron-EDTA or iron-DTPA to the microsomes. Catalase inhibited potently the oxidation of scavengers under all conditions, suggesting that H2O2 was the precursor of the hydroxyl radical in these systems. Very high amounts of superoxide dismutase had little effect on the iron-EDTA-stimulated rate of scavenger oxidation, whereas the iron-DTPA-stimulated rate was inhibited by 30 or 50% in microsomes or reductase, respectively. This suggests that the iron-EDTA and iron-DTPA chelates can be reduced directly by the reductase to the ferrous chelates, which subsequently interact with H2O2 in a Fenton-type reaction. Results with the reductase and microsomal systems should be contrasted with results found when the oxidation of hypoxanthine by xanthine oxidase was utilized to catalyze the production of hydroxyl radicals. In the xanthine oxidase system, ferric-ATP and -DTPA stimulated oxidation of scavengers by six- to eightfold, while ferric-EDTA stimulated 25-fold. Ferric-desferrioxamine consistently was inhibitory. Superoxide dismutase produced 79 to 86% inhibition in the absence or presence of iron, indicating an iron-catalyzed Haber-Weiss-type of reaction was responsible for oxidation of scavengers by the xanthine oxidase system. These results indicate that the ability of iron to promote hydroxyl radical production and the role that superoxide plays as a reductant of iron depends on the nature of the system as well as the chelating agent employed.
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PMID:The role of iron chelates in hydroxyl radical production by rat liver microsomes, NADPH-cytochrome P-450 reductase and xanthine oxidase. 633 21

Misonidazole, SR-2508, nitrofurazone and other nitroheterocycles stimulated release of 14CO2 from [1-14C]glucose but not from [6-14C]glucose when incubated with mouse Ehrlich ascites cells or human A549 lung carcinoma cells in vitro. This demonstrated that the nitro compounds activated the hexose monophosphate shunt and is evidence that an important pathway of nitro reduction in these cell lines is electron transfer from NADPH-dependent cytochrome c reductase to the nitro group. Shunt activity was stimulated under both aerobic and anaerobic conditions. For catalase-free Ehrlich cells, aerobic effects were greater than anaerobic, indicating that NADPH was used for reduction of H2O2, via GSH peroxidase and reductase, as well as for one-electron nitro reduction, under aerobic conditions. Several of the compounds tested stimulated 14CO2 release from [2-14C]glucose as well as from [1-14C]-glucose. This shows that the cellular requirement for NADPH, in the presence of nitro drug, was great enough to cause recycling of pentose phosphates. Recycling could decrease the availability of ribose-5-P needed for nucleic acid synthesis, which could partly explain the inhibition of DNA synthesis observed upon prolonged aerobic incubation of cells with nitro compounds. Comparison of the rate of disappearance of nitrofurazone from anaerobic A549 cell suspensions with the rate of 14CO2 release suggests that the drug reduction in this cell line was catalyzed almost entirely by NADPH-requiring enzymes.
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PMID:Nitroheterocycle metabolism in mammalian cells. Stimulation of the hexose monophosphate shunt. 642 13

Male mice were fed a diet containing less than 0.01 ppm selenium (Se-) for 6 months. A control group received the same diet containing 0.5 ppm selenium (Se+). In the livers of the Se- animals a drastic decrease in glutathione peroxidase (GSH-Px) activity was observed. It reached undetectable levels after 17 days of the Se- diet. At that time, GSH-transferase activity began to increase significantly, followed by changes in many other enzyme activities. After the 60th day, these enzyme modulations had reached a plateau with the following percentage changes compared to controls: GSH-transferases: 320% (1,2-dichloro-4-nitrobenzene), 218% (1-chloro-2,4-dinitrobenzene); glutathione reductase: 160%; ethoxycoumarin deethylase: 330%; cytochrome P-450-hydroperoxidase: 230%; heme oxygenase: 240%; UDP-glucuronyltransferase: 200%; GSH-thioltransferase: 64%; sulphotransferase: 62%; NADPH-cytochrome-P-450-reductase: 65%; flavin-containing mono-oxygenase: 57%. No significant changes were observed for GSH-transferase activity assayed with ethacrynic acid or for microsomal H2O2 formation and aniline hydroxylase activity. In single-pulse repletion experiments by injection of 250 micrograms selenium/kg body wt, different individual time constants for the recovery process of the enzymatic perturbations were observed. The half-times for the recovery ranged from 5.7 hr for the microsomal NADPH-cytochrome-P-450 reductase to over 29 hr for GSH-Px up to 44 hr for part of the GSH-transferase activity. 250 micrograms selenium/kg body wt were needed to restore 50% of GSH-Px activity in the long-term Se- mice compared to Se+ controls. All other enzymatic changes in the Se- mice needed a dose of 7 micrograms selenium/kg body wt for 50% restorage . The results demonstrate that processes other than those related to GSH-Px take place in a later phase of selenium deficiency in mouse liver with a chronologically common beginning. The different repletion and depletion kinetics as well as the different need of these processes for the trace element are discussed with respect to the existence of two separate selenium pools.
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PMID:Selenium and drug metabolism--II. Independence of glutathione peroxidase and reversibility of hepatic enzyme modulations in deficient mice. 642 18

We have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu,Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 microgram/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of cytosolic superoxide dismutase as a stimulator in anthranilamide hydroxylation by a microsomal monooxygenase system in rat liver. 644 2

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