SEARCH · PubMed Health

Results for “Luciferases, Firefly”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1998

Is there a specific receptor for anesthetics? Contrary effects of alcohols and fatty acids on phase transition and bioluminescence of firefly luciferase.

Firefly luciferase emits a burst of light when mixed with ATP and luciferin (L) in the presence of oxygen. This study compared the effects of long-chain n-alcohols (1-decanol to 1-octadecanol) and fatty acids (decanoic to octadecanoic acids) on firefly luciferase. Fatty acids were stronger inhibitors of firefly luciferase than n-alcohols. Myristyl alcohol inhibited the light intensity by 50% (IC50) at 13.6 microM, whereas the IC50 of myristic acid was 0.68 microM. According to the Meyer-Overton rule, fatty acids are approximately 12,000-fold stronger inhibitors than corresponding alcohols. The Lineweaver-Burk plot showed that myristic acid inhibited firefly luciferase in competition with luciferin, whereas myristyl alcohol inhibited it noncompetitively. The differential scanning calorimetry (DSC) showed that an irreversible thermal transition occurred at approximately 39 degrees C with a transition DeltaHcal of 1.57 cal g-1. The ligand effects on the transition were evaluated by the temperature where the irreversible change is half completed. Alcohols decreased whereas fatty acids increased the thermal transition temperature of firefly luciferase. Koshland's transition-state theory (Science. 1963. 142:1533-1541) states that ligands that bind to the substrate-recognition sites induce the enzyme at a transition state, which is more stabilized than the native state against thermal perturbation. The long-chain fatty acids bound to the luciferin recognition site and stabilized the protein conformation at the transition state, which resisted thermal denaturation. Eyring's unfolding theory (Science. 1966. 154:1609-1613) postulates that anesthetics and alcohols bind nonspecifically to interfacial areas of proteins and reversibly unfold the conformation. The present results showed that alcohols do not compete with luciferin and inhibit firefly luciferase nonspecifically by unfolding the protein. Fatty acids are receptor binders and stabilize the protein conformation at the transition state.

Alcohols↗

PubMed2000

The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen in a two-step process. The enzyme first catalyzes the adenylation of the carboxylate substrate luciferin with Mg-ATP followed by the oxidation of the acyl-adenylate to the light-emitting oxyluciferin product. The beetle luciferases are members of a large family of nonbioluminescent proteins that catalyze reactions of ATP with carboxylate substrates to form acyl-adenylates. Formation of the luciferase-luciferyl-AMP complex is a specific example of the chemistry common to this enzyme family. Site-directed mutants at positions Lys529, Thr343, and His245 were studied to determine the effects of the amino acid changes at these positions on the individual luciferase-catalyzed adenylation and oxidation reactions. The results suggest that Lys529 is a critical residue for effective substrate orientation and that it provides favorable polar interactions important for transition state stabilization leading to efficient adenylate production. These findings as well as those with the Thr343 and His245 mutants are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

Adenosine Monophosphate↗

PubMed2003

A mutagenesis study of the putative luciferin binding site residues of firefly luciferase.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed [Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319] a molecular graphics-based working model of the luciferase active site starting with the first X-ray structure [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] of the enzyme without bound substrates. In our model, the luciferin binding site contains 15 residues that are within 5 A of the substrate. Using site-directed mutagenesis, we made changes at all of these residues and report here the characterization of the corresponding expressed and purified proteins. Of the 15 residues studied, 12 had a significantly (>or=4-fold K(m) difference) altered binding affinity for luciferin and seven residues, spanning the primary sequence region Arg218-Ala348, had substantially (>or=30 nm) red-shifted bioluminescence emission maxima when mutated. We report here an interpretation of the roles of the mutated residues in substrate binding and bioluminescence color determination. The results of this study generally substantiate the accuracy of our model and provide the foundation for future experiments designed to alter the substrate specificity of firefly luciferase.

Animals↗

PubMed1986

Efficient singlet oxygen inactivation of firefly luciferase.

Firefly luciferase is inactivated by singlet oxygen at near diffusion controlled rates, 1.9 X 10(9) M-1 s-1, based on direct comparison with the oxidation of L-histidine. The inactivation kinetics are multiphasic. Inactivation is inhibitable by NaN3. Surface-separated-sensitizer (SSS) system in which singlet oxygen is produced above an air gap separating the reaction solution from the Rose Bengal sensitizer, ensuring only Type II reactions, was compared with a Sensitox II system in which the polymer bound Rose Bengal is contained in the reaction solution and both Type I and Type II reactions can occur. A slight stabilization is afforded by MgSO4.

Histidine↗

PubMed1993

Thermodynamics of anesthetic/protein interactions. Temperature studies on firefly luciferase.

Firefly luciferase is a soluble enzyme which is unusually sensitive to general anesthetics. The inhibition of the highly purified enzyme by three inhalational and three alcohol general anesthetics has been studied as a function of temperature, in the range from 5 to 20 degrees C. Inhibition constants Ki were determined at different temperatures, and van't Hoff plots of ln (Ki) versus reciprocal absolute temperature were found to be linear for all agents. Analysis of these plots gave values for the standard Gibbs free energy, enthalpy and entropy changes for transferring each anesthetic from water to the anesthetic-binding pocket on the protein. The most striking finding was that the enthalpy changes were much more negative for anesthetics binding to the protein than for binding to lipids or simple solvents. Furthermore, amongst the set of anesthetics studied, it was found that increasing potency correlated with favorable enthalpy rather than entropy changes. We discuss our results with respect to the molecular mechanisms underlying general anesthesia.

Anesthesia, General↗

PubMed2001

Cooperative binding of inhaled anesthetics and ATP to firefly luciferase.

Firefly luciferase is considered a reasonable model of in vivo anesthetic targets despite being destabilized by anesthetics, as reflected by differential scanning calorimetry (DSC). We examined the interaction between two inhaled anesthetics, ATP, luciferase, and temperature, using amide hydrogen exchange, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromoform cause destabilization, as measured by hydrogen exchange, suggesting nonspecific interactions. In the presence of ATP/Mg2+ and at room temperature, the anesthetics produce considerable stabilization with a negative DeltaH, indicating population of a conformer with a specific anesthetic binding site. Stabilizing interactions are lost, however, at unfolding temperatures. We suggest that preferential binding to aggregated forms of luciferase explain the higher temperature destabilization detected with DSC. Our results demonstrate a cooperative binding equilibrium between native ligands and anesthetics, suggesting that similar interactions could underlie actions at biologically relevant targets.

Adenosine Triphosphate↗

PubMedDate not supplied

Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice.

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents.

Animals↗

PubMedDate not supplied

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases.

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.

Amino Acid Sequence↗

PubMed2004

Identification of luciferyl adenylate and luciferyl coenzyme a synthesized by firefly luciferase.

The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP-HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light-production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low-oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase-catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl-coenzyme A synthetase.

Adenosine Triphosphate↗

PubMed1996

[Measurement of ATP in intact Escherichia coli cells, containing recombinant firefly luciferase].

Recombinant firefly luciferase was expressed in E. coli and its properties inside the intact cells were studied. At low concentrations, antibiotic polymyxin B increases permeability of E. coli cell membrane and concentrations of luciferase substrates inside the cells can thus be varied. Effect of intracellular ATP concentrations on intensity of bioluminescence was studied. Recombinant cells expressing the firefly luciferase gene can be used for investigation of substances which influence synthesis and hydrolysis of ATP inside the cells and cell membrane transport.

Adenosine Triphosphate↗

PubMed2001

The role of active site residue arginine 218 in firefly luciferase bioluminescence.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the Km values for luciferin. The contrasting near-normal Km values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

Amino Acid Sequence↗

PubMed2005

Identification of paralogous genes of firefly luciferase in the Japanese firefly, Luciola cruciata.

Two homologous genes of firefly luciferase, LcLL1 and LcLL2, were cloned from the Japanese firefly Luciola cruciata, and were expressed and characterized. The gene product of LcLL1 had long-chain fatty acyl-CoA synthetic activity, but not luciferase activity. The other gene product of LcLL2 did not show enzymatic activities of acyl-CoA synthetase and luciferase. RT-PCR analysis showed that the transcript of LcLL1 was abundant in larva but very low in adult, while LcLL2 was expressed in both larva and adult. Phylogenetic analysis indicated that LcLL1 and LcLL2 are paralogous genes of firefly luciferase. Recently, we found that CG6178 in Drosophila melanogaster is an orthologue of firefly luciferase and shows fatty acyl-CoA synthetic activity, but not luciferase activity. These results suggest that firefly luciferase might be evolved from a fatty acyl-CoA synthetase by gene duplication in insects.

Amino Acid Sequence↗

PubMed1998

Do anesthetics act by competitive binding to specific receptors? Phase transition of firefly luciferase.

1. Firefly luciferase (FFL) emits a flash of light when mixed with ATP and luciferin in the presence of molecular oxygen. 2. Halothane inhibited FFL at 0.22 mM, while myristic acid inhibited it at 0.68 microM. Under steady-state conditions, myristic acid competed with luciferin. The Lineweaver-Burk plots of anesthetics were nonlinear. 3. The Hill numbers for anesthetics were above 1, while that of myristic acid was below 1 (0.89). Anesthetics interact with FFL cooperatively at multiple sites. The binding site of myristic acid is limited. 4. FFL undergoes a phase transition at 37-40 degrees C. Anesthetics decreased, while myristic acid increased the transition temperature. 5. The contrasting effects of anesthetics and myristic acid are discussed according to Koshland's transition-state model for myristic acid, which involves specific receptors, and Eyring's unfolded-state model for anesthetics, which involves nonspecific conformational change. Fatty acids are receptor-binders and structure-maker of FFL. Anesthetics are non-specific binders and structure-breaker of FFL.

Acylation↗

PubMed2006

The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase.

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary diffraction studies of firefly luciferase from Photinus pyralis.

Firefly luciferase is a 62 kDa molecular weight enzyme which catalyzes a light-emitting reaction. Crystals of Photinus pyralis luciferase have been obtained by the microbatch technique, using polyethylene glycol as a precipitating agent. Firefly luciferase crystallizes as long needles which belong to the tetragonal space group P4(1)2(1)2, with cell dimensions a = 119.5, b = 119.5, c = 95.4 A. One molecule is present in the asymmetric unit. Diffracted intensities beyond 2.0 A resolution have been measured from frozen crystals using synchrotron radiation.

Journal Article↗

PubMed1999

Specific and non-specific binding of long-chain fatty acids to firefly luciferase: cutoff at octanoate.

Firefly luciferase emits a burst of light when the substrates luciferin and ATP are mixed in the presence of oxygen. We (I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052-1057) reported that long-chain fatty acids are specific inhibitors of firefly luciferase in competition with luciferin in microM ranges. They increased the thermal transition temperature. In contrast, 1-alkanols of the same carbon chain length inhibited the enzyme non-competitively in mM ranges and decreased the transition temperature. The present study showed that the action of fatty acids switched from specific to non-specific when the carbon chain length was reduced below C8 (octanoate). The fatty acids longer than C10 inhibited the enzyme in microM ranges whereas those shorter than C8 required mM ranges to inhibit it. The longer fatty acids increased whereas shorter fatty acids decreased the transition temperature. The Hill coefficients of longer chain bindings were less than one whereas those of shorter chain were more than one. The shorter fatty acids interacted with the enzyme cooperatively at multiple sites. Binding of the longer fatty acids is limited. Fatty acids longer than C10 are high-affinity specific binders and followed Koshland's induced-fit model. Those shorter than C8 are low-affinity non-specific denaturants and followed Eyring's rate process model. These results contradict the general consensus that the size of the receptor cavity discriminates specific binders.

Animals↗

PubMed2004

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases.

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases.

Animals↗

PubMedDate not supplied

Bioluminescence determination of enzyme activity of firefly luciferase in the presence of pesticides.

Firefly luciferase (EC 1.13.12.5) (FL) is the key enzyme in the firefly bioluminescence method (FB), which is widely used to determine the viability of living cells. The FB method can also be applied to monitoring the influence of different pollutants, such as pesticides. Firefly luciferase is a hydrophobic enzyme and its activity depends on the type of solvent, pH and substances present in the reaction mixture. The influence of three aromatic pesticides, including fenoxaprop-p-ethyl (I), diclofop-methyl (II) and metsulfuron methyl (III), on the enzyme activity was indirectly evaluated through the measurement of emitted light in the bioluminescence reaction, expressed in relative luminescence units (RLU). The reaction mixture used in the bioluminescence measurements consisted of: Tris buffer (pH 7.75), adenosine triphosphate (ATP) and ATP monitoring reagent, where FL is present. Ethanol-water solutions of each pesticide were then added at concentrations of 2.4 x 10(-4)-2.4 x 10(-8) mol/L. The FL activity inhibition factors (FL In%) were determined. The FL activity was maximally inhibited in the presence of all pesticides under study at a concentration of 2.4 x 10(-4) mol/L and was lowered by about 15-26% for pesticide I at concentrations of 2.4 x 10(-5)-2.4 x 10(-8) mol/L, whereas pesticides II and III, applied in the same concentration range, showed smaller FL inhibition values (5.3-20%). The pesticide degradation products (obtained after a 1 month period), measured in the same experimental conditions, in most cases exhibited a much less inhibitory effect on the enzyme activity than the corresponding initial pesticide.

Environmental Monitoring↗