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PubMed2005

Firefly luciferase enzyme fragment complementation for imaging in cells and living animals.

We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein-protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting d-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1-475)/Cfluc (245-550), Nfluc (1-475)/Cfluc (265-550), and Nfluc (1-475)/Cfluc (300-550). The Cfluc (265-550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1-475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering TAT-Cfluc (265-550) to cells stably expressing Nfluc (1-475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell-cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation.

3T3-L1 Cells↗

PubMed2003

The p53-inhibitor pifithrin-alpha inhibits firefly luciferase activity in vivo and in vitro.

BACKGROUND: Pifithrin-alpha is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-alpha in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-alpha provides a useful tool in the laboratory to investigate the function of p53 in model systems. RESULTS: While investigating the effects of pifithrin-alpha on the transcriptional activity of NF-kappaB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-alpha was observed both in vivo and in vitro. Pifithrin-alpha did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-alpha to active extracts inhibited this activity. Furthermore, pifithrin-alpha also inhibited recombinant firefly luciferase protein activity. CONCLUSIONS: Among its other biological activities, pifithrin-alpha is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.

Animals↗

PubMed2006

Catalytic properties of domain-exchanged chimeric proteins between firefly luciferase and Drosophila fatty Acyl-CoA synthetase CG6178.

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.

Adenine↗

PubMed1994

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase.

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.

Adenosine Triphosphate↗

PubMed1990

Targeting of cloned firefly luciferase to yeast mitochondria.

The firefly luciferase gene (luc) was fused to a 5' fragment of the 70-kDa protein gene (70K) from yeast. The fragment codes for the N-terminal putative signal sequence which targets and anchors the 70-kDa protein to the cytoplasmic side of the outer membrane in mitochondria. Two versions of the fusion gene, 70K[232]::luc and 70K[93]::luc (containing 292 and 93 5' codons from 70K, respectively), were constructed in a bacterial expression plasmid. Both the genes were expressed in Escherichia coli, and in both cases, bioluminescence activity was associated with the expression. The 70K[93]::luc gene was transferred to a yeast-bacteria shuttle vector used to transform Saccharomyces cerevisiae cells. As a control, the same strain was transformed with a plasmid including the original luc. With both transformants, bioluminescence activity was detected in intact cells and crude extracts. Upon growth on a nonfermentable carbon source and fractionation, the product of the fusion gene was associated mostly with mitochondria. In the control transformant, the product of luc was more delocalized. However, a significant amount remained associated with isolated mitochondria. No such spontaneous association of purified luciferase with wild-type mitochondria was observed in vitro. Trypsin treatment of mitochondria isolated from both transformed strains indicated that the fusion protein is anchored to the outer membrane and exposed to the medium while the unfused luciferase retained with the mitochondria is occluded in a compartment unaccessible to trypsin and released in the presence of detergent. The fusion protein retained the major catalytic properties of the parent firefly luciferase, as determined in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMedDate not supplied

Monitoring of spatial expression of firefly luciferase in transformed zebrafish.

The firefly luciferase gene attached to the cytomegalovirus promoter was transferred into zebrafish (Brachydanio rerio) by microinjection of fertilized eggs. Light emission could be monitored in vivo in eggs and throughout the early development of the fish by low-light video-image analysis. Gene expression was transient in most of the cases lasting for about 2 weeks. This gene cassette proved to be a very convenient and non-destructive transformation marker and the firefly luciferase gene appears to be a powerful tool for real-time imaging of tissue-specific gene expression in transgenic fish.

Animals↗

PubMed1994

Ethanol unfolds firefly luciferase while competitive inhibitors antagonize unfolding: DSC and FTIR analyses.

Firefly luciferase has gained popularity as a protein model in elucidating anaesthesia mechanism because the bioluminescence of the purified enzyme system is extremely sensitive to volatile anaesthetics. This study analysed the thermal unfolding of firefly luciferase by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). DSC showed that the transition of firefly luciferase from the folded (N) to unfolded (D) state occurred at 41.7 degrees C with the excess heat flow of 1.6 cal g-1 protein. Ethanol decreased the transition temperature dose dependently. In contrast, luciferin competitors, anilinonaphthalenesulphonate (ANS), toluidinonaphthalenesulphonate (TNS), and myristic acid increased the transition temperature. The competitive inhibitors antagonized unfolding and stabilized the N-state. Ethanol promoted unfolding and stabilized the D-state. Temperature scan by FTIR agreed with the DSC data. The intensities of amide-I' and amide-II' bands started to increase at 20-25 degrees C. This temperature coincides with the temperature where the bioluminescence of firefly luciferase is maximal. The unfolding effect of ethanol was evident even at 5 degrees C. ANS, TNS, and myristic acid completely protected the enzyme from the thermal unfolding. This is the first demonstration that the noncompetitive inhibitors induce the isothermal first-order phase transition in a functional protein, whereas competitive inhibitors protect the enzyme from thermal unfolding. The action mode of competitive inhibitors on firefly luciferase is completely different from that of noncompetitive inhibitors.

Calorimetry, Differential Scanning↗

PubMed2006

Cloning and characterization of the homologous genes of firefly luciferase in the mealworm beetle, Tenebrio molitor.

Three homologous genes of firefly luciferase were cloned from the non-luminous beetle Tenebrio molitor. Three gene products for homologues, TmLL-1, TmLL-2 and TmLL-3, showed fatty acyl-coenzyme A (acyl-CoA) synthetic activity, but not luciferase activity with firefly luciferin. The transcripts were detected through the developmental stages in T. molitor. These results suggested that firefly luciferase was evolved from a fatty acyl-coenzyme A synthetase by gene duplications in the insect.

Amino Acid Sequence↗

PubMed2003

One-step measurement of firefly luciferase activity in yeast.

Firefly luciferase is often used as a sensitive genetic reporter in various cell types. The pitfall in yeast, however, has been the need to break down the rigid cells in order to measure the enzyme activity. In this study we have removed the peroxisomal targeting codons from the Photinus pyralis luciferase gene (luc) and shown that in the yeast Saccharomyces cerevisiae this modified luciferase gives high levels of light emission that is easy to measure from intact living cells. Furthermore, cells with the modified luciferase grew essentially faster than those with the wild-type luciferase, indicating that peroxisomal targeting of a foreign enzyme puts some constraints to cellular viability. As a model system we used two different reporter constructs. In the first, expression of the luciferase gene is under control of CUP1-promoter, a well known yeast promoter that is inducible by copper ions. In the second, luciferase activity is dependent on activation of the human oestrogen receptor and its interaction with oestrogen-responsive elements incorporated in a yeast promoter. The luciferase activity measurement could be done on a 96-well plate by simple addition of the substrate, D-luciferin, at a moderately acidic pH of 5.0. The ease of use of the non-peroxisomal luciferase makes it an interesting alternative for reporter genes that are conventionally used in yeast, such as lacZ.

Copper↗

PubMed1996

Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes.

BACKGROUND: Firefly luciferase is a 62 kDa protein that catalyzes the production of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that utilize ATP in adenylation reactions. RESULTS: We have determined the crystal structure of firefly luciferase at 2.0 A resolution. The protein is folded into two compact domains. The large N-terminal domain consists of a beta-barrel and two beta-sheets. The sheets are flanked by alpha-helices to form an alphabetaalphabetaalpha five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft. CONCLUSIONS: Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.

Adenosine↗

PubMed1998

Structural basis for the inhibition of firefly luciferase by a general anesthetic.

The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-A resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.

Adenosine Triphosphate↗

PubMed2000

Development of a destabilized firefly luciferase enzyme for measurement of gene expression.

Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated, the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.

Animals↗

PubMed2004

Synthesis of dinucleoside tetraphosphates in transfected cells by a firefly luciferase reporter gene.

The firefly luciferase gene is widely used as a reporter gene and its expression is generally considered to be non-toxic. In addition to its light-producing reaction, luciferase can synthesise dinucleoside polyphosphates, intracellular signalling molecules, in vitro. Here we show that COS-7 cells transfected with a luciferase expression vector accumulate up to 0.5 mM adenine-containing dinucleoside tetraphosphates (Ap4N) during the 24 h following luciferin addition. The optimal external concentration of luciferin was 0.4-0.6 mM. In agreement with its poor ability to synthesise adenine-containing dinucleoside triphosphates in vitro, the level of these compounds did not increase after transfection. Consequently, the results of experiments involving luciferase-mediated light production by live cells should now be viewed in the light of the possible effects of an increased intracellular Ap4N concentration on the properties of the system under investigation. This observation also points to a useful non-invasive procedure for the specific enhancement of intracellular Ap4N for studies directed at understanding the functions of these compounds.

Animals↗

PubMed1987

Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase.

The gene for firefly luciferase (luc) can be used as a generalized genetic probe. A method that aids in the analysis of shuttle vectors containing luc by allowing verification in Escherichia coli of a functional coding sequence is presented here. Colonies containing a functional form of luc are detected on film after luciferin is added to initiate the luminescent reaction. Two conditions, lowering the pH of the environment and maintaining aerobic conditions, were found to greatly improve the sensitivity of the assay. This technique may be useful in the development of genetic constructs that alter the natural coding sequence of luc, such as in gene fusions.

Animals↗

PubMed2005

Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-CoA synthetase.

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.

Acyl Coenzyme A↗

PubMed1998

The N-terminal amino acid sequences of the firefly luciferase are important for the stability of the enzyme.

The structural and catalytic role of the N-terminal amino acid sequence of Photinus pyralis luciferase was investigated by site-directed mutagenesis. The firefly luciferase activity of a series of deletion and site-directed mutants in the amino-terminal region was investigated in vitro and in vivo. The mutant luciferases were produced either by in vitro transcription and translation or by expressing the cDNA encoding firefly luciferase in the yeast Saccharomyces cerevisiae as a fusion protein to the galactose DNA binding domain protein. Deleting the N-terminal amino acid residues from 3 to 10 dramatically reduced the luciferase activity to less than 1% of the wild-type activity. A marked decrease in the activity (to < 5%) was also observed with the Lys8Glu mutant. The Gly10Arg and Pro11Ala mutants showed 20-30% activity compared to the wild type. On the other hand, mutant Asn6Lys retained the wild-type activity level. Randomizing 3-11 N-terminal amino acids also showed a marked decrease in activity (< 1%). The mutant luciferases with extremely low levels of enzymatic activity were thermally unstable. These mutational and stability data correlate with the crystal structure of firefly luciferase in which specific amino acids in the N-terminal region form hydrogen bonds to the amino acids in the neighboring beta-stranded sheet. Because the N-terminal region is not part of the active site, the present results suggest that the highly conserved N-terminal amino acid sequences of the firefly luciferase are important for stabilizing the protein in its proper conformation for optimal enzyme activity.

Amino Acid Sequence↗

PubMed1994

Effect of periodate-oxidized ATP and other nucleotides on firefly luciferase.

Addition of periodate-oxidized ATP (oATP) to firefly luciferase-containing reaction mixtures enhanced light production when the reaction mixture contained > approximately 8 microM ATP. The time course of light production was changed from a flash pattern to a constant light output during incubations of < approximately 10 min. During longer incubation, firefly luciferase was inactivated in a concentration-dependent fashion by oATP. Firefly luciferase has two different time courses of light production that depend on ATP concentration (DeLuca and McElroy, Biochem. Biophys. Res. Commun., 123, 764, 1984). The enhancement of light production occurred only when higher ATP concentrations (> 8 microM) were used. There is little effect of oATP on firefly luciferase activity at low ATP concentrations (< 2 microM) which gave steady production of light. ATP did not antagonize the inactivation of firefly luciferase by oATP. When the oATP was chemically reduced with sodium borohydride (giving or ATP), there was no inactivation of firefly luciferase on incubation. When or ATP was used in a short incubation the enhancement of light production and time course change were the same as those observed with oATP. The corresponding AMP and adenosine compounds (o and or) were slightly inhibitory to firefly luciferase activity. ADP was without effect but both oADP and or ADP enhanced light production. Of these periodate-oxidized ADP, AMP, and adenosine derivatives only oADP inactivated firefly luciferase. The activating effect can be explained by a change in the conformation of the enzyme-product complex so that the product is released faster. In addition there is an inactivation of the enzyme by certain periodate-oxidized nucleotides during longer incubations.

Adenine Nucleotides↗

PubMed1992

Enhancement of firefly luciferase activity by cytidine nucleotides.

The temporal pattern of light production by firefly luciferase depends on the ATP concentration. With low concentrations of ATP a constant production of light occurred while at high concentrations of ATP (greater than 10 microM) there was a flash of light followed by a decline in light production. This time course of light production with high ATP concentrations was changed from the flash pattern to a pattern with a constant production of light by several cytidine nucleotides. CTP, CDP, dCTP, dCDP, dideoxyCTP, periodate-oxidized CTP and CDP, and the etheno derivatives of CTP and CDP produced that change. CMP, cytidine, CDP-glycerol, CDP-glucose, CDP-ethanolamine, and benzoylbenzoylCTP either were inhibitory to firefly luciferase or were not effective in changing the flash time course. Coenzyme A and related compounds also changed the time course of light production. The changes in time course produced by either cytidine nucleotides or CoA were inhibited by desulfoCoA. These compounds apparently enhanced light production by promoting the dissociation of the inhibitory product, oxidized luciferin, from the enzyme. When the activating compounds were used with high concentrations of ATP, the sensitivity of assay for firefly luciferase was increased. This increased sensitivity is important when using the firefly luciferase gene as a reporter.

Adenine Nucleotides↗