Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzymatic method for the determination of free glutamic acid in meat products and dried soups was collaboratively studied in 11 laboratories. In the presence of the enzyme glutamate dehydrogenase, L-glutamic acid is oxidatively deaminated by nicotinamide adenine dinucleotide (NAD) to 2-oxoglutarate. In a reaction catalyzed by diaphorase, the NADH thus formed converts 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride to a formazan, which is measured in the visible range at 492 nm. Fourteen samples (7 samples of minced sausage and 7 samples of dried cauliflower soup) with glutamate contents varying between 0.4 and 16 g/kg were included in the study. Materials were distributed to participants as blind duplicates and as split level pairs. The mean relative standard deviation (RSDR) for reproducibility for the dried soup material containing glutamate between 7 and 16 g/kg was 4.6%. RSDR values for samples of minced sausage containing glutamate at lower levels (0.4-1.3 g/kg) were between 12 and 16%.
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PMID:Enzymatic determination of free glutamic acid in dried soups and in minced sausages: NMKL collaborative study. 168 80

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active-site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gln. Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild-type enzyme, whereas considerable changes were observed in the spectra of the two-electron-reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro451----Ala [corrected] showed the greatest deviation from wild type. The enzyme is very easily over-reduced to the four-electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis-trans conversion. From studies on the pH dependence of the thiolate charge-transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450-mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450----Phe, -Ser and -Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge-transfer thiols. The difference in apparent pKa2 values between the His450-mutated enzymes is explained by changes in micropolarity. At the EH2 level the wild-type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852-862]. Based on the results obtained with the His450-mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild-type enzyme at the EH2 level is presented which takes three pKa values into account. The results of the pH dependence of the electronic species at the EH2 level of Glu455-mutated enzymes essentially follow the model proposed for the wild-type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge-transfer species.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Spectral properties of wild type and mutated enzymes. 168 37

Neutrophil myeloperoxidase, hydrogen peroxide, and chloride constitute a potent antimicrobial system with multiple effects on microbial cytoplasmic membranes. Among these is inhibition of succinate-dependent respiration mediated, principally, through inactivation of succinate dehydrogenase. Succinate-dependent respiration is inhibited at rates that correlate with loss of microbial viability, suggesting that loss of respiration might contribute to the microbicidal event. Because respiration in Escherichia coli can be mediated by dehydrogenases other than succinate dehydrogenase, the effects of the myeloperoxidase system on other membrane dehydrogenases were evaluated by histochemical activity stains of electrophoretically separated membrane proteins. Two bands of succinate dehydrogenase activity proved the most susceptible to inactivation with complete loss of staining activity within 20 min, under the conditions employed. A group with intermediate susceptibility, consisting of lactate, malate, glycerol-3-phosphate, and dihydroorotate dehydrogenases as well as three bands of glucose-6-phosphate dehydrogenase, was almost completely inactivated within 30 min. The relatively resistant group, including the dehydrogenases for glutamate, NADH, and NADPH and the remaining bands of glucose-6-phosphate dehydrogenase, retained substantial amounts of diaphorase activity for up to 60 min of incubation with the myeloperoxidase system. The differential effects of myeloperoxidase on dehydrogenase inactivation could not be correlated with published enzyme contents of flavin or iron-sulfur centers, potential targets of myeloperoxidase-derived oxidants. Despite the relative resistance of NADH dehydrogenase/diaphorase activity to myeloperoxidase-mediated inactivation, electron transport particles prepared from E. coli incubated for 20 min with the myeloperoxidase system lost 55% of their NADH oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential inactivation of Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated antimicrobial system. 169 36

The activity of oxidoreductases in the hippocampal formation of young adult (3-month old) and aged (24-month old) Wistar rats was compared by histochemical methods. A decreased activity of NADH-diaphorase and dehydrogenases involved in the Krebs cycle and glycolysis as well as an increased activity of glucose-6-phosphate dehydrogenase were demonstrated in the hippocampal nerve cells of aged rats. The activity of oxidoreductases in the glial cells of aged rats was increased. Degenerative changes in scattered mitochondria were seen ultrastructurally. No significant differences in the relative volume fraction of mitochondria in the presynaptic axon terminals and dendrites were found between young adult and aged rats.
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PMID:Age-related abnormalities of oxidoreductase activity and mitochondria in rat hippocampal formation. 169 44

1. After immunization of BALB/c mouse, four monoclonal antibodies against soluble NADH diaphorase from ejaculated boar spermatozoa were produced and characterized. The monoclonal antibodies were designated as follows Mab 1F2, Mab 4E2, Mab 5B8, Mab 5D8. 2. These monoclonal antibodies react with other enzyme forms-sedimentary NADH and NADPH and soluble NADPH and inhibit (although not completely) their activity. It is supposed that different forms of the enzyme share some common epitopes. 3. Treatment of ejaculated boar semen with 2O-methylcholanthrene causes an increase of the activity of the soluble diaphorase form only. 4. These results lead to the assumption that the sperm diaphorase is a dynamic enzyme system consisting of four immunologically similar isoenzymes although their functions are different.
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PMID:Characterization of NAD(P)H diaphorase from boar spermatozoa using specific monoclonal antibodies. 170 1

Specimens of the seawater fish annular seabream (Diplodus annularis) were caught from a polluted harbor area and from a clean reference area. Seawater concentrates and fish-muscle extracts were not mutagenic in the Salmonella reversion test. Liver preparations of fish from the 2 sources were comparatively assayed for microsomal mixed-function oxidases and cytosolic biochemical parameters, as well as for the ability of S12 fractions to activate promutagens or to detoxify direct-acting mutagens. A shift of the cytochrome P-450 peak from 450.3 to 448.5 was accompanied by a 4.5-fold increase in arylhydrocarbon hydroxylase activity in fish living in the polluted environment. At the same time, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were doubled in the cytosol of the same animals, while reduced glutathione (GSH) peroxidase and GSH S-transferase were slightly yet significantly depressed. No significant difference was recorded for other biochemical parameters, including GSH, oxidized glutathione (GSSG) reductase, NADH- and NADPH-dependent diaphorases, and DT diaphorase. In parallel, fish exposed to polluted seawater exhibited a significant and marked enhancement of the metabolic activation of the pyrolysis product Trp-P-2 and of benzo[a]pyrene-trans-7,8-diol, and at the same time were less efficient in detoxifying the antitumor compound ICR 191. Liver S12 fractions from both sources efficiently decreased the direct mutagenicity of sodium dichromate, and failed to activate benzo[a]pyrene and aflatoxin B1 to mutagenic metabolites. These results provide evidence that both biochemical parameters and the overall capacity of fish liver to activate or detoxify certain mutagens can be assumed to be sensitive indicators of exposure to mixed organic pollutants in the marine environment.
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PMID:Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater. 170 59

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.
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PMID:Cloning, sequence and overexpression of NADH peroxidase from Streptococcus faecalis 10C1. Structural relationship with the flavoprotein disulfide reductases. 171 12

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.
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PMID:Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775. 173 Feb 30

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain. 175 96

A two-step excisional treatment of a port-wine stain (PWS) on the back of a 43-yr-old female patient was performed. Immediately before the first surgical treatment, two corresponding series of argon laser impacts were performed, each on one PWS half. Different laser parameters with irradiances ranging from 95 to 382 W/cm2 and energy fluences ranging from 19 to 114,6 J/cm2 were used. Laser spots on the first part ot be excised were biopsied 10 min after laser treatment and prepared for histochemical analysis by staining with nitro blue tetrazolium chloride (NBTC). Reduction of this redox dye by nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase) leads on frozen tissue sections to an intense blue precipitate. The activity of NADH-diaphorase subsides immediately upon cell damage. All vital epidermal and dermal cells presented a dense blue granular pigment in their cytoplasm, sparing the nuclei. Laser induced arc-shaped epidermal and dermal necrosis did not stain, showing a clear demarcation from surrounding vital tissue. The depth of the thermal injury ranged from 0.28 to 0.45 mm; it did not correlate with the chosen fluences. With these penetration depths, the vast majority of PWS vessels was affected. Assessment of the remaining part of the PWS 8 months later yielded blanching of all laser-treated areas. With the NBTC method, an accurate definition of laser-induced tissue damage is feasible. It could be shown that the exposure time is the most relevant parameter influencing the penetration depth.
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PMID:Histochemical evaluation of the coagulation depth after argon laser impact on a port-wine stain. 175 55


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