Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous results from this laboratory demonstrated that glutathione concentrations decrease in aging mouse tissues. In this investigation glutathione (GSH) peroxidase and glutathione (GSSG) reductase activities were measured in tissues of standardized aging mice. Methods were validated for the quantitative determination of both enzymes in liver, kidney and heart tissues. GSH peroxidase activities were 27-53% lower in liver, kidney and heart of very old (36 months) mice compared to mature (10 months) mice (P less than 0.01). The same aging decreases were found with either hydrogen peroxide or cumene hydroperoxide as substrate. In a similar way GSSG reductase activities in liver and kidney were 25-28% lower in the old versus mature mice (P less than 0.01), but heart levels were unchanged. Further the lower GSSG reductase levels were unaffected by FAD supplementation in vitro. The changes in specific activity for both enzymes were not due to changes in organ weights and total protein contents, which were constant from 10 to 36 months of age. These decreases in GSH peroxidase and GSSG reductase do not account for the lower GSH levels in aging. Of special importance, however, is that these decreases indicate that detoxification via glutathione peroxidase and glutathione reductase could be impaired in senescence.
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PMID:Glutathione peroxidase and reductase activities in the aging mouse. 398 84

Human glutathione reductase (NADPH + GSSG + H+ in equilibrium with NADP+ + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD greater than 8-azido-FAD = 8-F-FAD = 8-C1-FAD greater than 4-thio-FAD = 1-deaza-FAD greater than 2-thio-FAD. The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD X glutathione reductase was crystallized for X-ray diffraction analysis. Detailed studies were focussed on position 8 of the flavin. 8-Cl-FAD X glutathione reductase and 8-F-FAD X glutathione reductase reacted only poorly with HS- to give 8-mercapto-FAD X glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. Other experiments showed that position 8 is accessible to certain solvent-borne reagents. 8-Mercapto-FAD X glutathione reductase, for instance, reacted readily and stoichiometrically with the thiol reagent methylmethanethiosulfonate. 8-Mercapto-FAD X glutathione reductase does not exhibit a long wavelength charge transfer absorption band upon reduction, as it is the case for the 2-electron-reduced FAD-containing enzyme. This behaviour indicates that the charge transfer interaction between flavin and the thiolate of Cys63 in the native enzyme is not per se essential for catalysis. The absorption spectrum of the blue anionic 8-mercapto-FAD bound to glutathione reductase suggests that the protein concurs to the stabilization of a negative charge in the pyrimidine subnucleus. In light of the protein structure this effect is attributed to the dipole moment of alpha-helix 338-354 which starts out close to the N(1)/C(2)/O(2 alpha) region of the flavin. 1-Deaza-FAD binds as tightly as FAD to the apoenzyme. The resulting holoenzyme was found to be enzymically active but structurally unstable. In this respect 1-deaza-FAD . glutathione reductase mimics the properties of the enzyme species found in inborn glutathione reductase deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:FAD analogues as prosthetic groups of human glutathione reductase. Properties of the modified enzyme species and comparisons with the active site structure. 398 92

Covalent modification of glutathione reductase (GR) from yeast with 1-fluoro-2,4-dinitrobenzene (FDNB) inhibited the NADPH-GSSG reductase activity completely. This modification also decreased the NADPH-thio-NADP+ transhydrogenase activity, stimulated the NADPH-oxidase activity, and induced the NADPH-cytochrome c reductase activity. Spectrophotometric titration showed that one tyrosine residue per FAD was modified with a dinitrophenyl group. The modified enzyme showed conversion of the two-electron reduced form (EH2) to the four-electron reduced form (EH4) in anaerobic conditions and conversion of EH2 to the oxidized form (E) in aerobic conditions. These results indicate that the modification of one tyrosine residue of the active site induces the instability of EH2.
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PMID:Effect of dinitrophenyl modification on oxidation-reduction of glutathione reductase from yeast. 403 Jul 49

Male weanling rats, fed a riboflavin-deficient diet for 14 days showed impairments in reactivity to the hyperphagic action of either insulin or 2-deoxy-D-glucose (2DG) and in sensitivity to the diabetogenic action of streptozotocin or alloxan. The intraperitoneal injection of riboflavin (160 micrograms/rat) resulted in an immediate restoration in FAD-dependent activation of erythrocyte glutathione reductase and in the reactivity of food intake to insulin, whereas the reactivity of food intake to 2DG was restored after 3 days of riboflavin repletion. The sensitivity to diabetogenic agents was not restored solely by the riboflavin injection but required 3 hours of feeding as well. These findings indicate that the riboflavin deficiency caused some defects at specific glucosensitive sites localized in the pancreas and the brain and that some metabolic processes were necessary to restore the sensitivity.
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PMID:Recovery from impairment in feeding response to glucoprivic stimuli and in sensitivity to diabetogenic agents in riboflavin-repleted rats. 622 19

The flavoprotein mercuric reductase catalyzes the two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. It has now been purified from Pseudomonas aeruginosa PAO9501 carrying the plasmid pVS1. In this plasmid system, where the mer operon is on the transposon Tn501, mercuric reductase comprises up to 6% of the soluble cellular protein upon induction with mercurials. The purification is a rapid (two-step), high yield (80%) procedure. Anaerobic titrations of mercuric reductase with dithionite revealed the formation of a charge transfer complex with an absorbance maximum around 540 nm. Striking spectroscopic similarities to lipoamide dehydrogenase and glutathione reductase were observed. These two enzymes, which catalyze the transfer of electrons between pyridine nucleotides and disulfides, are flavoproteins which contain an oxidation-reduction-active cysteine residue at the active site. The expectation that mercuric reductase contains a similar electron acceptor was confirmed when it was shown that mercuric reductase has the capacity to accept four electrons per FAD-containing subunit, and that two thiols become kinetically titrable by 5,5'-dithiobis-(2-nitrobenzoate) upon reduction with NADPH. These are characteristic features of the disulfide reductase class of flavoproteins. Further similarities with at least one of these enzymes, lipoamide dehydrogenase, include the E/EH2 midpoint potential (-269 mV), fluorescence properties, and extinction coefficients of E and EH2. Preliminary observations relevant to an understanding of the mechanism of mercuric reductase are discussed.
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PMID:Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide. 627

The nucleotide sequence of a 1980-base-pair segment of DNA, containing the lpd gene encoding the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method. The lpd structural gene comprises 1419 base pairs (473 codons, excluding the initiating AUG codon). It is preceded by a good promoter and an excellent ribosome binding site and it ends with a typical rho-independent terminator sequence. The results confirm that the lpd gene is an independent gene linked to, but not part of, the ace operon that encodes the E1 and E2 components of the pyruvate dehydrogenase complex. The location and transcriptional polarity of the lpd gene relative to the restriction map of the corresponding region of DNA, are completely consistent with previous genetic and post-infection labelling studies. The composition, Mr (50554 or 51274 if the FAD cofactor is included), amino-terminal sequence and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with previous studies on the purified enzyme. The enzyme also exhibits a remarkable degree of sequence homology with peptides of the pig heart enzyme and with other pyridine nucleotide disulphide oxidoreductases whose sequences have been defined: human erythrocyte glutathione reductase and plasmid-encoded mercuric reductase.
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PMID:Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12. 635 60

The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2'.5'-ADP-Sepharose affinity column from which it was specifically eluted by a 0-10 mM NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells, 6.9 mg of pure enzyme was obtained after a 2632-fold purification, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein, with an A272/A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 A hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH, respectively, as reductants. Apparent K'm values of 16, 377, and 66 microM were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD, NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing significantly only after 10 min at 70 degrees C. A marked activity loss was observed however, even at 0 degrees C, in the presence of 20 microM NADPH. The enzyme was inactivated by low concentrations of para-hydroximercuribenzoate; the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.
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PMID:Purification by affinity chromatography of glutathione reductase (EC 1.6.4.2) from Escherichia coli and characterization of such enzyme. 639 25

The amino acid sequence of ferredoxin-NADP+ oxidoreductase [EC 1.18.1.2, FNR] from Spirulina sp., a blue-green alga, was determined. Spirulina ferredoxin-NADP+ oxidoreductase was composed of 294 amino acid residues and the molecular weight of the holoenzyme was 34,135. An apparent homology of the amino(N)-terminal region was found between ferredoxin-NADP+ reductases from Spirulina and spinach. We also found some sequence similarities in human erythrocyte glutathione reductase and p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, both of which are NADPH-dependent FAD enzymes.
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PMID:Spirulina ferredoxin-NADP+ reductase. The complete amino acid sequence. 643 Aug 89

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.
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PMID:Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide. 643 80

The elucidation of the primary structure of the Escherichia coli lipoamide dehydrogenase (EC 1.8.1.4) by sequencing the corresponding structural gene (lpd) has enabled a detailed structural comparison between lipoamide dehydrogenase and the related disulphide oxido-reductase, human erythrocyte glutathione reductase (EC 1.6.4.2). Some 28% of the amino acid residues were found to be identical and a striking degree of homology was apparent throughout the polypeptide chains. It was concluded that the two enzymes possess very similar three-dimensional structures with particularly strong conservation of residues around the FAD and NAD(P) binding sites and at the redox centres of the molecules. Significant amino acid substitutions occur in the substrate binding pocket and these include an extra 18 amino acid residues at the C terminus of lipoamide dehydrogenase. Under physiological conditions, lipoamide dehydrogenase and glutathione reductase act in opposite directions, passing reducing equivalents to NAD+ or from NADPH (respectively), and two key substitutions near the redox centre could be associated with this difference in function. This study represents the first direct structural comparison between two related enzymes that are NADP+-linked (glutathione reductase) and NAD+-linked (lipoamide dehydrogenase). The differential recognition of these two cofactors could be explained in terms of amino acid substitutions. A divergent evolutionary relationship between the two enzymes including their NAD and NADP binding domains is fully supported by this analysis.
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PMID:Structural relationship between glutathione reductase and lipoamide dehydrogenase. 654 54


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