Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The luciferase of the bioluminescent boring mollusc, Pholas dactylus, has been purified by a new method which includes centrifugation in cesium chloride gradients. Homogeneous preparations have been obtained and molecular weight determinations and subunit analysis support the idea that this preparation is an oxyluciferin-luciferase complex. The preparation catalyzes oxidation of ascorbic acid in presence of H2O2, and this peroxidase activitity has been used for characterization (thermal and pH stabilities, activity as a function of pH, isoelectric point, turnover number). The existence of two atoms of copper has been established and their involvement in the peroxidase activity indicated. Chemical analyses have shown that Pholad luciferase is a glycoprotein and the existence of glucosamine, fucose, mannose, and galactose residues has been demonstrated. The apparent buoyant dentisty (1.340), the sedimentation coefficient (10.7 S), the Stoke's radius (83 A), the partial specific volum (0.707), and the molecular weight (350,000) have been determined. The frictional ratio (flf0 = 1.8) derived from the Stoke's radius indicates that the molecule is asymmetric. The quaternary structure has been examined. Subunits of molecular weight 150,000 and 46,000 have been observed. The latter has electrophoretic properties identical with luciferin or oxyluciferin.
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PMID:Characterization and properties of Pholas luciferase as a metalloglycoprotein. 23 92

The identity and stoichiometry of the reaction products of the oxygenated reduced flavin bacterial luciferase intermediate isolated by Sephadex chromatography at low temperature have been determined under two conditions, allowing the reaction to go to completion by warming either in the presence or absence of long chain aliphatic aldehyde. In the latter case, very little bioluminescence occurs, and 1 mol each of H2O2 and FMN is produced per mol of enzyme intermediate. In the presence of aldehyde, the formation of an aldehyde-enzyme intermediate complex can be detected by optical absorption spectroscopy at -30 degrees; upon warming, bioluminescence with high quantum yield occurs with the formation of 1 mol of FMN but no H2O2.
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PMID:The oxygenated bacterial luciferase-flavin intermediate. Reaction products via the light and dark pathways. 80 33

Bacterial luciferase from Photobacterium phosohoreum was found to produce bioluminescence on reaction with FMN and H2O2 in the presence of aldehyde. This luminescence is presumably produced by the same X1 intermediate as that found in FMNH2-O2 initiated luminescence. From the ratio of the light intensities of the FMN-H2O2 initiated reactions, we calculated the association constant of the reaction, luciferase+FMN+H2O2in equilibriumX1, and estimated its temperature dependence. From these results we calculated the thermodynamic parameters of the reaction, luciferase+FMNH2+O2in equilibriumX1. We found that the free energy level of X1 is only 3.2 kcal below that of fr-e FMN and H2O2. We also estimated the thermodynamic parameters of other steps of the luminescent reaction. The values obtained showed that the formation of X1 from luciferase, FMNH2 and O2 involves a positive entropy change, but the intermediate is in a state stabilized against decomposition. Results also suggest a considerable degree of electronic rearrangement on formation of the excited-state molecule from the X1-aldehyde complex.
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PMID:Studies on luciferase from Photobacterium phosphoreum. VIII. FMN-H2O2 initiated bioluminescence and the thermodynamics of the elementary steps of the luciferase reaction. 95 Mar 35

The solution chemistry of N(5)-alkyl flavinium cations and radical species formed by their le- reduction are discussed. Previously unknown, the 4a-flavine hydroperoxides are established to be formed on reaction of N(5)-alkyl flavinium cations with H2O2 or on reaction of N(5)-alkyl-1, 5-dihydroflavines with 3O2. The stability of the 4a-flavine hydroperoxide species is exemplified in the isolation and characterization of 4a-hydroperoxy-N(5)-ethyl-3-methyl-lumiflavine. 4a-Flavine hydroperoxide compounds are shown to be stronger oxidants than H2O2, and to undergo a chemiluminescent reaction in the presence of an aldehyde. Preliminary observations on the chemiluminescent reaction of 4a-flavine hydroperoxides + RCHO are provided, and these are compared to those in the literature dealing with the bioluminescence of bacterial luciferase in the presence of 3O2 and RCHO.
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PMID:Simple synthesis of a 4a-hydroperoxy adduct of a 1,5-dihydroflavine: preliminary studies of a model for bacterial luciferase. 106 19

The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses.
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PMID:Stopped-flow kinetic analysis of the bacterial luciferase reaction. 156 36

Toxic oxygen species have been implicated as important mediators of injury after reperfusion of an ischemic organ. The aim of this study was to determine if prior metabolic inhibition, such as that which occurs during ischemia, potentiates oxidant injury in vitro. Bovine pulmonary artery endothelial cells were metabolically inhibited for various periods of time with or without the mitochondrial inhibitor oligomycin (650 nM). The cells were rescued from metabolic inhibition by a wash step and subsequent addition of 5.5 mM glucose. At the same time that metabolic inhibition was relieved the cells were subjected to doses of H2O2 ranging from 0 to 100 microM. ATP levels were monitored over a 2-hr time course after rescue from metabolic inhibition by the luciferin-luciferase assay. Cell viability at 2 hr after relief of metabolic inhibition was assessed by trypan blue exclusion. Intracellular pH during metabolic inhibition was determined with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein tetraacetomethoxymethyl ester. H2O2 consumption, a measure of H2O2 scavenging capability, was determined by a fluorescent assay. The viability and ATP levels of cells not subjected to metabolic inhibition were unaffected by these low concentrations of H2O2. Cells metabolically inhibited with glucose depletion and oligomycin were exquisitely sensitive to H2O2. Cells that were only deprived of glucose demonstrated no potentiation of injury, while cells subjected to mitochondrial inhibition with oligomycin alone also showed significant potentiation of oxidant injury. H2O2 consumption was not affected by metabolic inhibition. Conditions associated with mitochondrial inhibition consistently resulted in a decrease in intracellular pH. These experiments suggest that a synergism exists between metabolic inhibition and subsequent oxidant exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Metabolic inhibition potentiates oxidant injury. 202 Jan 85

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H2O2 (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.
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PMID:Coupled reactions for the determination of analytes and enzymes based on the use of luminescence. 280 Dec 29

In the bacterial luciferase reaction, light emission is due to the mixed function oxidation of FMNH2 and long chain aldehydes, which leads to the formation of an electronically excited product species, postulated to be luciferase-bound 4a-hydroxy flavin. In the present work it was found that H2O2 stimulates an additional and kinetically distinct luminescence. The stimulation is more apparent in reactions inhibited by long chain alcohols, and the H2O2 is effective even if added secondarily. The stimulation requires H2O2 only at the outset; its subsequent destruction by catalase does not diminish the response, appreciably.
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PMID:Enhancement of light emission in the bacterial luciferase reaction by H2O2. 357 Dec 7

Asbestos causes persistent increases in c-jun mRNA and AP-1 DNA binding activity in hamster tracheal epithelial (HTE) cells, the progenitor cell type of asbestos-induced bronchogenic carcinoma. Studies here were designed to determine mechanisms of c-jun induction by asbestos and the phenotypic consequences of Jun expression in HTE cells. To examine whether asbestos or H2O2 induced transcription of c-jun, we transiently transfected HTE cells with a plasmid containing a fragment of the c-jun promoter coupled to a luciferase reporter gene. In addition, c-jun was overexpressed in cells using a full-length human c-jun construct, and effects on proliferation and transformation were examined. HTE cells transfected with the jun-luciferase construct showed increased luciferase activity when exposed to crocidolite asbestos or H2O2. These results demonstrate that asbestos and H2O2 activate AP-1-dependent gene transcription. Overexpression of c-jun led to increased proliferation and enhanced ability of HTE cells to grow in soft agar, an indication of cellular transformation. Data suggest that overexpression of c-jun may contribute to asbestos and oxidant-induced proliferation and carcinogenesis.
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PMID:Transcriptional activation of the proto-oncogene c-jun by asbestos and H2O2 is directly related to increased proliferation and transformation of tracheal epithelial cells. 779 93

Cultured rat glomerular mesangial and epithelial cells and bovine glomerular endothelial cells were exposed to various concentrations of hydrogen peroxide (H2O2). Mesangial cells treated with 10 to 100 microM H2O2 for 24 hours showed a two- to ninefold increase in Mn-SOD mRNA expression associated with significantly (P < 0.005) increased Mn-SOD activity (22.2 +/- 1.2 and 12.2 +/- 0.7 mu/mg protein for H2O2 100 microM treated and untreated cells, respectively). In contrast, expression of Cu-Zn SOD and beta-actin mRNA was not affected by H2O2. Induction of Mn-SOD mRNA by H2O2 was inhibited by actinomycin-D (4 microM) treatment. Glomerular endothelial cells also showed an increase in Mn-SOD mRNA expression following 100 microM H2O2 treatment, as did glomerular epithelial cells following treatment with 500 and 1000 microM H2O2 but not with 100 microM. Transcriptional activity of the Mn-SOD gene was assessed with a fusion reporter gene consisting of a luciferase gene (pGL2P) and a 1.2 kb fragment from the rat Mn-SOD genomic DNA (-806 to +408 bp of the transcription initiation site, -806:+408). The construct was transfected into rat glomerular mesangial and epithelial cells. Mesangial and epithelial cells transfected with pGL2P (-806:+408) and treated with H2O2 (100 microM and 1 mM for mesangial and epithelial cells, respectively) demonstrated some threefold increase in luciferase activity, whereas cells transfected with pGL2P lacking the Mn-SOD fragment did not show changes in luciferase activity following H2O2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Oxidants induce transcriptional activation of manganese superoxide dismutase in glomerular cells. 796 52


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