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)

We developed a technique to map the distribution of selected metabolites in the growth cartilage in situ using luciferase-NAD(P)H:FMN oxidoreductase. Chick tibial epiphyses were freeze-trapped, sectioned, and freeze-dried. For evaluating lactate, luciferase was suspended in a buffer containing polyvinylalcohol, gelatin, NAD, FMN, and lactic dehydrogenase (LDH). The buffer was frozen into a layer 800 microns thick and placed in contact with the tissue section. The temperature of the frozen reagent mixture was then allowed to increase; the emitted light was focused through a photographic lens and collected on film. We found that lactate was synthesized by cells in all regions of the growth plate. The highest concentration of the metabolite was observed in the calcified hypertrophic region. Substantial levels of lactate were also present in articular cartilage. By modifying the composition of the buffer solution, we were able to map the distribution of glucose and glucose-6-phosphate and the activity of LDH. Maximal levels of each of the three components were present in hypertrophic cartilage. Chemical analysis of the tissue section confirmed the luminographic studies and provided further evidence that there was reliance on glycolytic metabolism in terminally differentiated chondrocytes. Use of enzyme couples similar to those described above should permit the technique to be used to study most, if not all, of the major metabolic components of cartilage.
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PMID:Measurement of metabolic events in the avian epiphyseal growth cartilage using a bioluminescence technique. 846 50

In principle, luminometry allows very sensitive metabolite measurements as shown with standards in aqueous solutions (e.g., buffers). However, components of complex biological samples may largely interfere with luminometric reactions. We now describe a procedure by which subnanomole amounts of intermediary metabolites (malate, glucose 6-phosphate) can be measured by luminometry in extracts from isolated mammalian cells, namely rat heart muscle cells. Basically, measurements occur in two steps: (i) Enzymatically catalyzed reactions involving the metabolite to be measured lead to the stoichiometric production of NAD(P)H; (ii) the oxidation of this NAD(P)H in a luciferase/reductase system results in light production which is proportional to the original concentration of the metabolite. The reaction scheme is thus as follows: (1) Metabolite (malate, glucose 6-phosphate) + NAD(P)+ --> X + NAD(P)H + H+; (2) NAD(P)H + O2 + RCOH --> NAD(P)+ + RCOOH + H2O + hnu. The cardiomyocytes used are previously subjected to an ethanolic extraction in which the cellular NAD(P)H is destroyed by acidification. Subsequent evaporation of the extracts allows to neutralize and to concentrate the samples. This contributes, along with other experimental maneuvers, to increasing the sensitivity of the method. With this procedure, we were able to detect amounts of approximately 70 pmol of malate and approximately 90 pmol of glucose 6-phosphate in cardiomyocyte samples. In addition, the calculated cellular concentrations of malate and glucose 6-phosphate (101.1 +/- 4.5, and 202.8 +/- 26.1 microM, respectively, in the absence of exogenous substrate) correspond to values previously reported for heart tissue. In principle, the procedure described could be applied to the measurement of any ethanol-extractable metabolite that can be converted in reactions involving NAD(P)+.
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PMID:Luminometric measurement of subnanomole amounts of key metabolites in extracts from isolated heart muscle cells. 866 Jun 23

Transposon-generated mutant strain UCD 328 of Nostoc punctiforme strain ATCC 29133 has a phenotype of an increased sensitivity to a hormogonium-inducing factor exuded by a symbiotic plant partner, Anthoceros punctatus, and an initial increased hormogonium-dependent infection of the plant. Sequence analysis showed that the transposition site in strain UCD 328 lies within a 1,251-bp open reading frame (ORF), designated hrmA, that displays no significant similarity to known database sequences. A second, 837-bp ORF (hrmU) ends 2 bp 5' from the start of hrmA and has the signature sequences belonging to a family of NAD(P)H-dependent oxidoreductases. Strains having insertional mutations in hrmU or hrmA reproduce the strain UCD 328 phenotype. Transcriptional fusions of luxAB to hrmU or hrmA show an 8- to 10-fold peak increase in luciferase activity 13 to 20 h after the start of incubation in the presence of an aqueous extract of A. punctatus. A promoter induced by the extract was deduced to be between 2.0 to 3.4 kb from the translational start of hrmU. A multicopy plasmid that contains hrmUA within a 6.2-kb fragment conferred an increased infection phenotype on wild-type N. punctiforme 29133. This plasmid and another plasmid containing 4.4 kb of DNA 5' of the transposition site prevented extract-dependent induction of hrmA-luxAB transcription in strain UCD 328, implicating titration of some trans-activator(s) by the cloned fragments. We hypothesize a role for hrmUA in the inhibition of hormogonium formation by the metabolism of an unknown hormogonium-regulating metabolite.
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PMID:A hormogonium regulating locus, hrmUA, of the cyanobacterium Nostoc punctiforme strain ATCC 29133 and its response to an extract of a symbiotic plant partner Anthoceros punctatus. 905 33

The mechanisms of reduced flavin transfer in biological systems are poorly understood at the present. The Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the luciferase pair were chosen as a model for the delineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to mediate the reduction of the flavin substrate by NADPH, exhibited a ping-pong kinetic pattern with a Km, FMN of 8 microM and a Km,NADPH of 20 microM in a single-enzyme spectrophotometric assay monitoring the NADPH oxidation. However, the kinetic mechanism of FRP was changed to a sequential pattern with a Km,FMN of 0.3 microM and a Km,NADPH of 0.02 microM in a luciferase-coupled assay measuring light emission. In contrast, the Photobacterium fischeri NAD(P)H-FMN oxidoreductase FRG showed the same ping-pong mechanism in both the single-enzyme spectrophotometric and the luciferase-coupled assays. Moreover, for the FRP, FMN at concentrations over 2 microM significantly inhibited the coupled reaction in both light intensity and quantum yield, and showed apparent noncompetitive and competitive inhibition patterns against NADPH and luciferase, respectively. No inhibition of the NADPH oxidation was detected under identical conditions. These results are consistent with a scheme that the reduced flavin cofactor of FRP is preferentially utilized by luciferase for light emission, the reduced flavin product generated by the reductase is primarily channeled into a dark oxidation, and luciferase competes against flavin substrate in reacting with the FRP reduced flavin cofactor. An FRP derivative containing 2-thioFMN as the cofactor was also used to further examine the mechanism of flavin transfer. Results again indicate a preferential utilization of the reductase reduced flavin cofactor by luciferase for the bioluminescence reaction.
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PMID:Mechanism of reduced flavin transfer from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase. 977 91

Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.
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PMID:Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. 1020 54

Bacterial bioluminescence, catalyzed by FMN:NAD(P)H oxidoreductase and luciferase, has been used as an analytical tool for quantitating the substrates of NAD(P)H-dependent enzymes. The development of inexpensive and sensitive biosensors based on bacterial bioluminescence would benefit from a method to immobilize the oxidoreductase and luciferase with high specific activity. Toward this end, oxidoreductase and luciferase were fused with a segment of biotin carboxy carrier protein and produced in Escherichia coli. The in vivo biotinylated luciferase and oxidoreductase were immobilized on avidin-conjugated agarose beads with little loss of activity. Coimmobilized enzymes had eight times higher bioluminescence activity than the free enzymes at low enzyme concentration and high NADH concentration. In addition, the immobilized enzymes were more stable than the free enzymes. This immobilization method is also useful to control enzyme orientation, which could increase the efficiency of sequentially operating enzymes like the oxidoreductase-luciferase system.
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PMID:Specific immobilization of in vivo biotinylated bacterial luciferase and FMN:NAD(P)H oxidoreductase. 1032 74

NAD(P)H-flavin oxidoreductases [flavin reductases (FR)] are a class of enzymes capable of producing reduced flavin for bacterial bioluminescence and other biological processes. Bacterial luciferase utilizes oxygen, reduced FMN (FMNH2) and a long-chain aliphatic aldehyde as substrates for light emission. The Vibrio harveyi luciferase and FRP (for which we have cloned the gene and determined the crystal structure) is a model for the elucidation of the reduced flavin transfer mechanism using both a flavin reductase single-enzyme assay monitoring the NADPH oxidation and a flavin reductase-luciferase coupled assay measuring bioluminescence intensity or quantum output. The FRP exhibits a ping-pong kinetic pattern in the single-enzyme assay but changes to a sequential pattern in the coupled assay. Furthermore, FMN at >2x10(-6) mol/L reduced both the light intensity and quantum yield of the coupled reaction by noncompetitively inhibiting NADPH and competitively inhibiting luciferase. These results support a scheme in which the luciferase forms specific complex(es) with FRP. Indeed, such complexes were shown by fluorescence anisotropy to exist between luciferase and monomeric FRP either in the holo- or apoenzyme form. Furthermore, the reduced flavin cofactor of FRP is transferred directly to luciferase for bioluminescence, whereas the reduced flavin product of FRP is inefficient in supporting the luminescence reaction. The mechanism of reduced flavin transfer is apparently flavin and flavin reductase specific.
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PMID:Probing the mechanisms of the biological intermolecular transfer of reduced flavin. 1072 98

It is believed that the reduced FMN substrate required by luciferase from luminous bacteria is provided in vivo by NAD(P)H-FMN oxidoreductases (flavin reductases). Our earlier kinetic study indicates a direct flavin cofactor transfer from Vibrio harveyi NADPH-preferring flavin reductase P (FRP(H)) to the luciferase (L(H)) from the same bacterium in the in vitro coupled luminescence reaction. Kinetic studies were carried out in this work to characterize coupled luminescence reactions using FRP(H) and the Vibrio fischeri NAD(P)H-utilizing flavin reductase G (FRG(F)) in combination with L(H) or luciferase from V. fischeri (L(F)). Comparisons of K(m) values of reductases for flavin and pyridine nucleotide substrates in single-enzyme and luciferase-coupled assays indicate a direct transfer of reduced flavin, in contrast to free diffusion, from reductase to luciferase by all enzyme couples tested. Kinetic mechanisms were determined for the FRG(F)-L(F) and FRP(H)-L(F) coupled reactions. For these two and the FRG(F)-L(H) coupled reactions, patterns of FMN inhibition and effects of replacement of the FMN cofactor of FRP(H) and FRG(F) by 2-thioFMN were also characterized. Similar to the FRP(H)-L(H) couple, direct cofactor transfer was detected for FRG(F)-L(F) and FRP(H)-L(F). In contrast, despite the structural similarities between FRG(F) and FRP(H) and between L(F) and L(H), direct flavin product transfer was observed for the FRG(F)-L(H) couple. The mechanism of reduced flavin transfer appears to be delicately controlled by both flavin reductase and luciferase in the couple rather than unilaterally by either enzyme species.
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PMID:Differential transfers of reduced flavin cofactor and product by bacterial flavin reductase to luciferase. 1132 36

The 11-cis retinol dehydrogenase (11-cis-RoDH) enzyme catalyzes the oxidation of cis-retinols to their respective retinals, a rate limiting step in the formation of retinoic acids. Earlier, we have shown that the enzyme also exhibits an oxidative 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) activity that can convert 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) into dihydrotestosterone (DHT), the most potent natural androgen. 11-cis-RoDH could thus control the formation of two active hormones, namely 9-cis retinoic acid and DHT. Therefore, depending upon the substrate availability in the various tissues, this enzyme could provide different metabolites for specific cell functions. To further investigate the role of 11-cis-RoDH in the formation of DHT from 3alpha-diol, we stably expressed the enzyme in the human embryonic kidney cell line 293 (HEK-293). The transformation of 3alpha-diol by these cells was evaluated by assays using both microsomal fractions and intact cultured cells stably expressing 11-cis-RoDH. The results show that in the intact cells 11-cis-RoDH only catalyzes the oxidation of 3alpha-diol into DHT whereas the microsomal fraction catalyzes both the oxidation and the reduction reactions depending upon whether NAD(+) or NADH is added. Furthermore, we examined the ability of 11-cis-RoDH, through the production from 3alpha-diol of the active androgen DHT, to activate the androgen-responsive promoter of the prostate-specific antigen (PSA) gene. The co-transfection of the pCMV expression vector containing 11-cis-RoDH (pCMV-11-cisRoDH), a luciferase reporter gene driven by a PSA promoter (pCMV-PSA-Luc) and an androgen receptor (pCMV-hAR) showed that, in the presence of 3alpha-diol, the expression of the PSA promoter is increased by five to six-fold. Moreover, this stimulatory effect is inhibited by hydroxyflutamide, a well-known antiandrogen. These results suggest that 11-cis-RoDH could be involved in a non-classical pathway of androgen formation and might play a role in the modulation of the androgenic response in some peripheral tissues.
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PMID:Modulation of the androgenic response by recombinant human 11-cis retinol dehydrogenase. 1137 78

Extracellular ATP is a widespread autocrine/paracrine signal since many animal cells release ATP in the extracellular medium; often this release is mechanosensitive, but the molecular mechanism is still unclear. The involvement of vesicular release, conductive channels, or ABC transporters has been suggested in different cell types. We investigated the mechanism of ATP release in human HOBIT osteoblastic cells, in which mechanical stimulation induced intercellular calcium waves sustained by both cell-to-cell coupling through gap junctions and ATP release. In this study we employed a luciferin-luciferase bioluminescence assay to measure the amount of ATP released under different stimulatory conditions. Given the role of connexin hemichannels in favoring passive NAD(+) transport [Bruzzone, S., et al. (2001) FASEB J. 15, 10-12], the involvement of connexin hemichannels as putative ATP transporters was initially investigated. In HOBIT cells overexpressing connexin43 the amount of nucleotide released under basal and stimulated conditions was similar to non-transfected cells, ruling out a major involvement of connexin hemichannels in ATP transport. In nontransfected HOBIT cells mechanical stimulations induced by medium displacement and hypotonic stress consistently enhanced ATP efflux. Cytochalsin D treatment did not alter basal and stimulated ATP release, while elevated cAMP levels consistently reduced efflux in both cases. ATP released by hypotonic stress and medium displacement evoked intracellular Ca(2+) transients in fura2-loaded HOBIT cells, indicating that different mechanical stimuli activate physiological cell responses.
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PMID:Mechanically induced ATP release from human osteoblastic cells. 1174 33


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