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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of different antioxidant pathways in cultured rat pleural mesothelial cells was studied by exposing the cells to various hydrogen peroxide (H2O2) concentrations and by measuring H2O2 cell cytotoxicity and the capacity of the cells to scavenge H2O2. The antioxidant enzymes, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and catalase were analyzed biochemically. Catalase and CuZn superoxide dismutase were localized by immunocytochemistry. To enable investigation of the glutathione redox cycle and catalase pathways, glutathione reductase was inactivated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and catalase was inactivated with aminotriazole. When the cells were exposed to a low, sublethal (0.030 mM) H2O2 concentration, glutathione reductase but not catalase inactivation resulted in a decreased capacity to remove H2O2 from the extracellular medium. When the cells were exposed to a high (0.25 mM) H2O2 concentration, H2O2-scavenging capacity decreased remarkably when catalase was inactivated. When the cells were exposed to 0.1 to 0.5 mM H2O2, cell cytotoxicity (lactate dehydrogenase release) increased significantly if glutathione reductase was inactivated; catalase inactivation resulted in a significant cytotoxicity only at high (greater than or equal to 0.25 mM) H2O2 concentrations. Immunocytochemical studies showed that the cells, both in situ and in vitro, contained low amounts of catalase. This suggests that the results of the catalase-inhibition studies are probably not due to a change in the characteristics of the cells in culture. 3-Aminobenzamide is a compound that is known to prevent NAD depletion through inhibition of poly(ADP-ribose) polymerase during oxidant stress. When intact cells were treated with different antioxidants and exposed to 0.5 mM H2O2, both catalase and 3-aminobenzamide protected the cells completely.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1992 Jul
PMID:Antioxidant defense mechanisms in cultured pleural mesothelial cells. 162 38

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.
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PMID:Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum. 164 69

DNA fragments encoding streptococcal NADH peroxidase (NPXase) have been amplified, cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The NPXase gene (npr) comprises 1341 base-pairs and is preceded by a typical ribosome binding site. Upstream from the structural gene, putative -10 and -35 promoter regions have been identified, as has a possible factor-independent terminator that occurs in 3'-flanking sequences. The deduced relative molecular mass (Mr = 49,551), amino acid composition and isoelectric point of NPXase are in good agreement with previous values obtained with the purified enzyme. In addition, three sequenced peptides totaling approximately 20% of the protein were located in the npr gene product. From the sequencing data the deduced NPXase sequence shares low but significant homology with the flavoprotein disulfide reductase class of enzymes ranging from 21% for glutathione reductase (GRase) to 28% for thioredoxin reductase. Alignment of NPXase to Escherichia coli GRase allowed the identification of three previously reported fingerprints for the FAD, NADP+ and central domains of GRase, in the peroxidase sequence. In addition, Cys42 of NPXase, which is present as an unusual stabilized cysteine-sulfenic acid in the oxidized enzyme, aligns favorably with the charge-transfer cysteine in E. coli GRase, and both residues closely follow FAD-binding folds found near their respective amino termini. Such sequence characteristics can also be seen in mercuric reductase, lipoamide dehydrogenase and trypanothione reductase, suggesting that all these enzymes may have originally diverged from a common ancestor. Sequences that are on average 50% identical with three previously reported peptides of the related streptococcal NADH oxidase were also identified in the NPXase primary structure, suggesting a strong similarity between these flavoenzymes. Using the E. coli phage T7 expression system the npr gene has now been overexpressed in an E. coli genetic background. The resultant overexpressing clone produced a recombinant NPXase that was catalytically active and immunoreactive to NPXase antisera.
J Mol Biol 1991 Oct 05
PMID:Cloning, sequence and overexpression of NADH peroxidase from Streptococcus faecalis 10C1. Structural relationship with the flavoprotein disulfide reductases. 171 12

In order to investigate the basis of functional diversity among the pyridine nucleotide-oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor- mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide-disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.
Mol Microbiol 1991 Jan
PMID:Molecular characterization of the gor gene encoding glutathione reductase from Pseudomonas aeruginosa: determinants of substrate specificity among pyridine nucleotide-disulphide oxidoreductases. 184 5

Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 microM EDTA or 10 microM DETAPAC yielding full protection. Ag+, Zn2+ and Cd2+ potentiated the redox inactivation promoted by NADPH alone, while Cr3+, Fe2+, Fe3+, Cu+, and Cu2+ protected the enzyme. The Zn2+ and Cd2+ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn2+ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn2+ or Cd2+. The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP+ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.
Mol Cell Biochem 1991 Mar 13
PMID:Metals are directly involved in the redox interconversion of Saccharomyces cerevisiae glutathione reductase. 186 75

The structure of lipoamide dehydrogenase from Azotobacter vinelandii has been refined by the molecular dynamics technique to an R-factor of 19.8% at 2.2 A resolution. In the final model, the root-mean-square deviation from ideality is 0.02 A for bond lengths and 3.2 degrees for bond angles. The asymmetric unit comprises two subunits, each consisting of 466 amino acid residues and the prosthetic group FAD, plus 512 solvent molecules. The last ten amino acid residues of both chains are not visible in the electron density distribution and they are probably disordered. The operation required to superimpose the two chains forming the dimer is a rotation of exactly 180 degrees with no translation component. The final model shows the two independently refined subunits to be very similar, except for six loops located at the surface of the molecule. The structure of each subunit of the enzyme consists of four domains with the catalytic centre located at the subunit interface. The reactive disulphide bridge, 48-53, is oxidized with S gamma of Cys53 located 3.5 A away from carbon C-4a of the isoalloxazine ring. The side-chain of His450' points its N epsilon 2 towards S gamma of Cys48 and is hydrogen bonded to the carboxylate of Glu455'. The FAD is bound in an extended conformation and the isoalloxazine ring is not completely planar with an angle between the pteridine and the benzene ring of 7.3 degrees in the first subunit and of 12.1 degrees in the second one. The overall folding of lipoamide dehydrogenase is very similar to that of glutathione reductase. However, a comparison of the two enzymes, which have only 26% sequence identity, reveals significant conformational differences. These concern the tertiary as well as the quaternary structure of the two molecules. In each subunit of lipoamide dehydrogenase the NAD-binding domain and the interface domain appear to be differently oriented with respect to the FAD-binding domain by 7.1 degrees and 7.8 degrees, respectively. The interface domain contains, in addition, major changes in tertiary structure. Furthermore, the two subunits forming the dimer appear to be shifted with respect to each other by more than 4 A, when the lipoamide dehydrogenase dimer is compared with that of glutathione reductase. In spite of all these changes at the tertiary and quaternary level the active sites of the enzymes, which occur at the dimer interface, appear to be remarkably similar.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1991 Aug 20
PMID:Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase. 188 Aug 7

The crystal structure of NADH peroxidase (EC 1.11.1.1) from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been refined to a resolution of 2.16 A using the simulated annealing method. The final crystallographic R-factor is 17.7% for all data in the resolution range 7 to 2.16 A. The standard deviations are 0.015 A in bond lengths and 3.0 degrees in bond angles for the final model, which includes all 447 amino acid residues, one FAD and 369 water molecules. The enzyme is a symmetrical tetramer with point group D2; the symmetry is crystallographic. The redox center of the enzyme consists of FAD and a cysteine (Cys42), which forms a sulfenic acid (Cys-SOH) in its oxidized state. A histidine (His10) close to Cys42 is likely to act as an active-site base. In the analyzed crystal, the enzyme was in a non-native oxidation state with Cys42 oxidized to a sulfonic acid Cys-SO3H. The chain fold of NADH peroxidase is similar to those of disulfide oxidoreductases. A comparison with glutathione reductase, a representative of this enzyme family, is given.
J Mol Biol 1991 Oct 20
PMID:Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 A resolution. 194 54

In seven rabbits subjected to suprarenal aortic coarctation hypertension, the segments above and below the coarctation were tested for the antioxidant defences (i.e. acid-soluble thiol compounds, selenium-dependent and selenium-independent glutathione peroxidase, glutathione reductase, glutathione transferase) and thiobarbituric acid-reactive substances. Seven sham-operated rabbits served as controls. Systolic blood pressure proximal to the ligature increased significantly with respect to pre-operative values after 16 days (117 +/- 8.3 vs 71.7 +/- 5.2 mmHg, P less than 0.05), while pressure distal to the ligature remained normotensive. Higher values of acid-soluble thiol compounds, thiobarbituric acid-reactive substances and increased activities of selenium-dependent glutathione peroxidase, glutathione reductase and glutathione transferase were assayed in the suprarenal with respect to the subrenal segment in both groups. However, the values of the upper segments were more elevated in the experimental group than in controls, but no differences were observed in the lower segments. Glutathione peroxidase activity assayed with cumene hydroperoxide was higher than the activity assayed with hydrogen peroxide in the hypertensive segments, but no differences were detected in the substenotic and control segments. Furthermore, an isoenzymatic form of glutathione transferase, analogous to rat 8-8 glutathione transferase isoenzyme, was detected by immunodiffusion in the hypertensive aorta. The following conclusions may be drawn: (1) a biochemical gradient in glutathione-related enzymes, acid-soluble thiol compounds and thiobarbituric acid-reactive substances between the proximal and distal aorta seems to exist in control rabbits; (2) suprarenal aortic coarctation induces a significant increase in glutathione-related antioxidant defences and thiobarbituric acid-reactive substances of the hypertensive aortic wall.
J Mol Cell Cardiol 1991 Jun
PMID:Aortic glutathione-related antioxidant defences in rabbits subjected to suprarenal aortic coarctation hypertension. 194 85

The effects of a subclinical fascioliasis at various stages of its development (at week 3, 6 and 9 after infection by oral administration of 20 metacercariae of Fasciola hepatica) in rats were determined on the activity of enzymes involved in liver metabolism of glutathione and on the subunit pattern of cytosolic glutathione S-transferase. The parasitic pathology was ascertained by clinical observation of the rats and at autopsy. Hepatic microsomal cytochrome P-450 content was significantly decreased in infected rats by week 3 and 6 post-infection. Not correlatively, the catalytic activities of glutathione S-transferase towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene were significantly lowered in last stages of the experimental fascioliasis (by week 6 and 9 post-infection). These decreases were correlated to that of subunit 1 as determined by means of high-performance liquid chromatography of cytosolic proteins whereas subunit 6 could also be decreased. Fascioliasis did not alter cytosolic glutathione, glutathione reductase and glutathione peroxidase activities or plasma glutathione S-transferase activity accepting 1-chloro-2,4-dinitrobenzene as the substrate.
Mol Biochem Parasitol 1991 Feb
PMID:Differential inhibition of rat hepatic glutathione S-transferase isoenzymes in the course of fascioliasis. 205 25

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na+ by oxidizing Na+-channels. Increased intracellular Na+ leads to an increase in Na+/Ca2+ exchange. The increased Na+ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca2+ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.
Mol Cell Biochem
PMID:The effect of oxidants on biomembranes and cellular metabolism. 251 41


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