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PubMed1983

Presence of octopamine in firefly photomotor neurons.

Various tissues involved in producing luminescence in larval fireflies (Photuris versicolor) were examined for the presence of octopamine. These tissues included the terminal abdominal ganglion (A8) which innervates the paired lantern organs, the cell bodies of the photomotor neurons and the isolated larval lanterns. A previous study has identified the 4 motoneurons arising from A8 which bilaterally innervate the paired larval lanterns through symmetrical axons existing both sides of the ganglion. Individual photomotor neuron somata were isolated, pooled and found to contain about 0.03 pmol/soma giving an effective concentration of 2.8 mM octopamine. Significant amounts of octopamine were also found within the peripheral effector tissue. The presence of octopamine throughout the luminescence-producing pathway further supports the hypothesis that octopamine serves a neurotransmitter function in firefly bioluminescence. In this system, it appears that octopamine serves a more direct role as a neurotransmitter that that postulated for its modulatory and hormonal functions in other arthropod systems. Furthermore, the bioluminescent response of the larval firefly lantern provides a useful dynamic system to study the physiology, pharmacology and biochemistry of octopaminergic transmission.

Animals↗

PubMed1999

Does pressure antagonize anesthesia? High-pressure stopped-flow study of firefly luciferase and anatomy of initial flash.

The antagonizing effect of high pressure against anesthesia is well known. With purified firefly luciferase, however,. Biophys. J. 60:1309-1314) reported that high pressure did not affect the initial flash intensity. Firefly luciferase emits a burst of light when the substrates luciferin and ATP are added in the presence of O2. The light intensity decays rapidly and the weak light lasts for hours. The initial flash is a transient event and is not in a steady state. The steady state is represented by the slope of the linear part of the integral of the light output. The present study used a high-pressure stopped-flow system to compare the pressure effects on the initial flash intensity and the steady-state light intensity. The flash intensity did not change by the application of hydrostatic pressure in the presence or absence of chloroform or 1-octanol. In contrast, high pressure increased the steady-state light intensity. The application of 12 MPa pressure increased the steady-state light intensity of firefly luciferase inhibited by 5 mM chloroform or 0.7 mM 1-octanol by 19.7% and 18.8%, respectively. When analyzed by the rapid reaction kinetics of the transition state theory, the initial peak intensity represents the total amount of active enzyme and is unrelated to the reaction rate. Anesthetics inhibited the initial flash by unfolding the protein, thereby decreasing the concentration of the active enzyme. Pressure affected the steady-state light intensity by changing the reaction rates.

Anesthesia↗

PubMed1999

Does pressure antagonize anesthesia? Opposite effects on specific and nonspecific inhibitors of firefly luciferase.

Ueda and Suzuki (1998. Biochim. Biophys. Acta. 1380:313-319; 1998. Biophys. J. 75:1052-1057) reported that myristic acid inhibited firefly luciferase in microM range in competition with luciferin, whereas anesthetics inhibited it in millimeter ranges noncompetitively with luciferin. Myristate increased, whereas anesthetics decreased, the thermal denaturation temperature. The present study showed that high pressure increased the steady-state light intensity of the halothane-doped firefly luciferase but decreased that of the myristate-doped firefly luciferase. The steady-state light intensity showed a maximum at 19.1 degrees C. At 19.1 degrees C, high pressure did not affect the light intensity in the absence of the inhibitors. In the presence of 0.5 mM halothane, however, 25 MPa pressure (maximum effect) increased the light intensity to 106.0% of the control without the inhibitor. In the presence of 2.5 microM myristate, 40 MPa pressure decreased the light intensity to 90.9% of the control. When the temperature was 25 degrees C in the absence of inhibitors, 40 MPa pressure increased the light intensity 119.2% of the ambient value. At 0.5 mM halothane, 40 MPa pressure further increased the light intensity to 106.1% above the control 40 MPa value. At 2.5 microM myristate, 40 MPa pressure decreased the light intensity to 90.1% of the control 40 MPa value. From the pressure dependence of the light intensity, the volume change DeltaV of the enzyme was estimated at 25 degrees C: 0.5 mM halothane increased DeltaV = +3.93 cm3 mol-1, whereas 2.5 microM myristate decreased DeltaV = -7.66 cm3 mol-1. Present results show that there are distinct differences between the specific and nonspecific ligands in their response to high pressure. Myristate, which competes with luciferin, decreased the protein volume and stabilized the conformation against thermal perturbation. Halothane, which does not compete with the substrate, increased the protein volume and destabilized the conformation.

Anesthesia↗

PubMed1997

Cloning and sequencing of a cDNA for firefly luciferase from Photuris pennsylvanica.

The first cDNA from the Photurinae subfamily of the Lampyridae encoding a firefly luciferase from lantern mRNA of Photuris pennsylvanica has been cloned, sequenced, the amino-acid sequence predicted and the sequence reported to GenBank. The cDNA was about 1.8 kb in length with the largest open reading frame coding for a 545-residue protein. The 5' noncoding region is 61 bp long and the 3' noncoding region is 135 bp in length. There is a 24-nucleotide poly(A) tail. When the amino-acid residues are aligned, P. pennsylvanica contains 154 (about 28% of the total residues) that are conserved in all 16 of the deduced luciferase sequences that are presently available. In this P. pennsylvanica luciferase, the amino acids at 276 of the positions are the same at corresponding positions of at least one of the other enzymes. There are two amino-acid differences between this luciferase and the unpublished sequence obtained by Dr. Keith Wood for a putative larval Photuris firefly luciferase cloned from a Maryland firefly. Signature amino-acid sequences and domains found in the deduced sequence are for adenylate kinase, the putative AMP-binding domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.

Amino Acid Sequence↗

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↗

PubMed1987

Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase.

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.

Adenosine Triphosphate↗

PubMed1998

Site-directed mutagenesis of histidine 245 in firefly luciferase: a proposed model of the active site.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We previously reported [Branchini, B. R., Magyar, R. A., Marcantonio, K. M., Newberry, K. J., Stroh, J. G., Hinz, L. K., and Murtiashaw, M. H. (1997) J. Biol. Chem. 272, 19359-19364] that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analogue, was a potent photoinactivation reagent for luciferase. We identified a luciferase peptide 244HHGF247, the degradation of which was directly correlated to the photooxidation process. We report here the construction and purification of wild-type and mutant luciferases H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins are consistent with His245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His245 is oxidized to aspartate during the photooxidation reaction was supported by the extremely low specific activity ( approximately 300-fold lower than WT) of the H245D mutant. Using the crystal structures of luciferase without substrates [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 [Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997) EMBO J. 16, 4174-4183] as a starting point, we have performed molecular-modeling studies and propose here a model for the luciferase active site with substrates luciferin and Mg-ATP bound. We have used this model to provide a structure-based interpretation of the role of 244HHGF247 in firefly bioluminescence.

Amino Acid Substitution↗

PubMed1977

Source of oxygen in the CO(2) produced in the bioluminescent oxidation of firefly luciferin.

Incorporation of (18)O into the CO(2) produced in the bioluminescent oxidation of firefly luciferin was studied. In H(2) (16)O medium with (18)O(2) gas, the product CO(2) contained up to 75% C(16)O(18)O, showing that one O of the product CO(2) arose from the O(2) that oxidized luciferin. This result is consistent with a dioxetane mechanism. Analysis of the mass spectral data of the CO(2) obtained in high-enrichment H(2) (18)O medium with (16)O(2) gas indicated the presence of about 20% contaminating CO(2), which contributes approximately 70% of the total incorporated (18)O. Thus the values of incorporated (18)O in H(2) (18)O medium with (16)O(2) gas have no significance in the present context. Data obtained with luciferases of the American firefly Photinus and Japanese firefly Luciola were similar.

Journal Article↗

PubMed1985

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.

Animals↗

PubMed1987

Firefly luciferase is targeted to peroxisomes in mammalian cells.

Although several enzymes known to reside in peroxisomes have been studied extensively, no cis-acting amino acid sequences involved in the transport of these proteins to peroxisomes have been described. As a first step towards the determination of a putative peroxisomal targeting sequence, we have expressed the cDNA encoding the firefly luciferase [Photinus-luciferin:oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] in monkey kidney cells and found that the product of the gene is transported to peroxisomes. Luciferase is derived from the firefly (Photinus pyralis) and is synthesized and stored in the cells of the firefly's lantern organ, where it is also found in peroxisomes. The fact that this protein is similarly targeted in cells from such different organisms suggests that the process of protein transport to peroxisomes has been highly conserved through evolution.

Animals↗

PubMed1996

Efficient folding of firefly luciferase after transport into mammalian microsomes in the absence of luminal chaperones and folding catalysts.

Folding of polypeptides emerging from the protein translocase in the membrane of mammalian microsomes was analyzed after synthesis of corresponding precursor proteins in a mammalian translation system. Firefly luciferase was used as a model protein; the corresponding hybrid precursor contained the preprolactin signal peptide. The rates and efficiencies of folding of luciferase in microsomes were compared with those of folding of luciferase in the cytosol. Furthermore, folding of luciferase in microsomes was compared with that in proteoliposomes, i.e. in the absence of luminal molecular chaperones and folding catalysts. Folding in microsomes was less efficient compared with folding in the cytosol. Folding in the absence of luminal proteins was more efficient compared with folding in their presence and identical to folding in the cytosol. Thus, firefly luciferase emerging from translocase can efficiently fold to its native conformation without chaperoning by any luminal proteins. There may be molecular chaperones present in the microsomal membrane that can efficiently substitute for the cytosolic chaperone machinery comprising Hsp40, Hsp60, and Hsp70 with respect to folding of firefly luciferase.

Adenosine Triphosphate↗

PubMed1967

The spectral distribution of firefly light. II.

The in vivo peak emission wavelengths of bioluminescence are reported for 15 species of American fireflies. A spectrophotometric study of the dorsal light organs of 155 specimens of the Jamaican firefly Pyrophorus plagiophthalamus showed three distinct color distributions with peak emission wavelengths at 550.1 +/- 1.5 mmicro, 556.8 +/- 1.4 mmicro, and 562.4 +/- 1.0 mmicro. Similar spectral measurements of 35 ventral light organs of the same insects gave peak emission wavelengths ranging from 547 through 594 mmicro. This is a wider distribution than the total range of all 34 species of firefly studied to date. There was no obvious correlation between the colors of the ventral and dorsal light organs. It appears that P. plagiophthalamus is a special case in which the luciferase enzyme is not only different among members of the same species, but it may be different for the dorsal and ventral light organs in a single individual. A minimum of six different luciferase molecules for P. plagiophthalamus ventral light organs is proposed. The statistical precision in making these spectrophotometric measurements is discussed.

Animals↗

PubMed1975

Limits of applicability of the firefly luminescence ATP assay for the detection of bacteria in clinical specimens.

ATP measurement can be used as an indicator of biological mass, and the extreme sensitivity of the firefly ATP assay has led to its use in bacterial detection systems. Clinical specimens present problems not encountered with cultured isolates of known bacterial species. The lower limit of sensitivity for detecting bacteria using the firefly assay is 100,000 bacteria per ml. Non-bacterial ATP, which is probably present in all clinical specimens, produces false-positive results unless it is completely destroyed, and this destruction must be carried out under conditions that do not affect bacterial ATP. A cause of false-negative results is the presence in all urine specimens of unidentified materials that inhibit the luminescent enzymic reaction. These considerations indicate that application of the firefly ATP assay in bacterial detection systems for clinical specimens is feasible only if a preparatory step separates bacteria from interfering materials and from non-bacterial sources of ATP, and concentrates microorganisms to measurable levels. These limitations sharply curtain the applicability in diagnostic microbiology of this exotic chemical reaction.

Adenosine Triphosphate↗

PubMed2000

Refolding of firefly luciferase immobilized on agarose beads.

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.

Animals↗

PubMed2005

Second-generation laser firefly clusters: improved scheme for distributed sensing in the atmosphere.

We present an improved concept of laser firefly clustering for atmospheric probing, elaborating on previous published work. The laser firefly cluster is a mobile, flexible, and versatile distributed-sensing system, whose purpose is to profile the chemical and the particulate composition of the atmosphere for pollution monitoring, meteorology, detection of contamination, etc. The fireflies are deployed in situ at the altitude of interest and evoke a backscatter response from aerosols and molecules in the immediate vicinity by using a coded laser signal. The enhanced, second-generation system affords better performance at lower energy cost and has a considerably increased scope of application. A numerical example demonstrates the potential of the innovative system.

Journal Article↗

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↗

PubMedDate not supplied

Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling.

Several reporter gene assays have been described where gene transcription is activated as a consequence of a specific signal transduction event, such as activation of adenylyl cyclase (1.2). Reporter genes typically consist of specific responsive elements placed upstream of a minimal promoter, which together control the expression of a readily detectable reporter protein, such as luciferase. We have developed a dual glow-signal firefly and Renilla luciferase assay, which allows the simultaneous measurement of two reporter genes in the same well of a 96-well plate. In this report we demonstrate the use of this assay for the simultaneous analysis of agonist activity at two G-protein coupled receptors which signal through activation of the G-protein alpha sub-unit, G alpha S. Chinese hamster ovary (CHO) cells stably transfected with a cAMP responsive firefly luciferase reporter were further transfected with the human Vasopressin V2 receptor. Similarly, CHO cells stably transfected with a cAMP responsive Renilla luciferase reporter were further transfected with the human beta 2-adrenoceptor. The two cell lines were mixed in individual wells of a 96-well plate and a number of compounds were screened to determine their activity at both receptors. Stimulation with vasopressin and beta 2-adrenoceptor agonists resulted in the activation of the firefly and Renilla luciferases respectively. Stimulation with forskolin, which directly stimulates adenylyl cyclase, caused the activation of both reporter genes, and stimulation with a range of further compounds with no activity at either receptor did not generate a reporter response. The dual luciferase assay allows the simultaneous screening of two receptors in a 96-well format resulting in significant time and cost savings.

Animals↗

PubMed1976

Kinetics of the light flash of the living firefly: a supramolecular process.

The light intensity vs. time curve of the light flash of the living firefly has been measured. Unlike the purified firefly enzyme system in aqueous solution, the living system does not show light decay conforming to a double exponential time curve, to simple first or second order decay, or to solid-state Elovich kinetics. Light decay of the living flash does show linearity in a probit vs. square root of time plot, which may indicate a reaction rate-limited by cooperative interactions of a biological phase transition. The observation that the kinetics of the firefly light system differ in the living cell from those in the purified system suggests that in the living system supramolecular factors control the rate of the reaction.

Animals↗