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PubMed2005

Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family.

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.

Cell-Free System↗

PubMed1996

Separation, identification and determination of luciferin in the Iranian firefly, Lampyris turkestanicus by HPLC and spectroscopic methods.

Iranian firefly larvae, Lampyris turkestanicus collected from north of Iran and their luciferin were analyzed by HPLC, TLC, MS and spectroscopic Fourier transform ([FT]-IR, FT-NMR, UV and fluorescence) methods. The results showed that luciferin in L turkestanicus lanterns was the same as in the American firefly, Photinus pyralis and synthetic D-luciferin.

Animals↗

PubMed1980

Localization of fluorescent compounds in the firefly light organ.

Two fluorescent materials have been localized in the adult firefly light organ by fluorescence microscopy. One of these is located in photocyte granules, has a maximum emission between 510 and 540 nm, is more fluorescent in basic than acidic solution, and is unstable in ultraviolet light, phosphomolybdic acid, and potassium permanganate. It is thought to be luciferin. The fluorescence of this material is very dim in untreated fireflies but increases substantially following sustained light emission induced by synephrine or prolonged electrical stimulation. It is suggested that the luciferin of untreated animals is bound in the granules and that binding suppresses its fluorescence. The second fluorescent material is located in the dorsal layer of the light organ, particularly in the cells bordering on the photogenic layer. This material has a maximum emission between 510 and 520 nm, is relatively stable in ultraviolet light, and rapidly disappears when light organs are exposed to water. Its identity and function are unknown.

Animals↗

PubMed2003

Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay.

We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03 x 10(-20) and 2.05 x 10(-20) mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.

Acetate Kinase↗

PubMed1999

Model of the active site of firefly luciferase.

A model for the spatial structure of firefly luciferase--ATP--luciferin complex is suggested using the coordinates of unliganded luciferase and the enzyme--substrate complex of the adenylating subunit of gramicidin S synthetase known from the literature. Conformational changes in luciferase can occur during substrate binding resulting in a relative orientation of two luciferase domains similar to that in case of the AMP--phenylalanine--synthetase complex. The model is consistent with data on the physicochemical properties of firefly luciferase and its complexes with the substrates.

Adenosine Triphosphate↗

PubMed1999

Two-site expression immunoassay using a firefly luciferase-coding DNA label.

BACKGROUND: We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase. METHODS: The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction. RESULTS: The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44). CONCLUSIONS: The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.

Animals↗

PubMed1983

Firefly and bacterial luminescence: basic science and applications.

The basic chemistry of the reactions leading to light emission in the firefly and in bacteria are briefly reviewed. With excess firefly reagents, the light intensity is proportional to the ATP concentration. For this reason, the reagents have been used for ATP determination in a number of important biological systems. A number of such applications are reviewed. With excess bacterial reagents, the light intensity is directly proportional to the reduced pyridine nucleotide concentration (NADH or NADPH). The applications of this system for studying reactions involving dehydrogenases using NAD or NADP as electron acceptors are presented. Many assays have now been developed using enzymes immobilized on Sepharose. The advantages of using the immobilized enzymes are greater stability of the immobilized enzymes over the soluble forms; increased sensitivity of detection relative to the soluble forms, and reusability of the immobilized enzymes. A comparison of the immobilized bioluminescent assay for 7 alpha-hydroxysteroid with gas-liquid chromatography and radioimmunoassay is presented. Coimmobilized enzymes can be packed in a flow cell and used in an automated instrument with good reproducibility. It is likely that future developments of bioluminescent assays for ATP or NAD(P)H will be with immobilized enzymes using an automated instrument.

Adenosine Triphosphate↗

PubMedDate not supplied

Use of firefly luciferase for ATP measurement: other nucleotides enhance turnover.

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (> 8 microM) or a fairly constant production of light with lower concentrations of ATP (< 1 microM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.

Adenine Nucleotides↗

PubMedDate not supplied

Immobilization of luciferase from a firefly lantern extract on glass strips as an alternative strategy for luminescent detection of ATP.

The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution, luciferase is unstable and cannot be re-used, a problem that can be partially circumvented by immobilizing the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilizing matrices, since it allows most of the emitted photons to be detected. We report a new method for luciferase immobilization on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation. Our method is based on the co-immobilization by adsorption of luciferase (from a firefly lantern extract) and poly-L-lysine (PL) on non-porous glass strips. Luciferase immobilized in this way exhibits minimal variations in intersample activity, high sensitivity for ATP detection (linear luminescence responses down to 50 nmol/L) and good stability (full activity for at least 60 days when stored at -80 degrees C). PL-mediated immobilization of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research.

Adenosine Triphosphate↗

PubMedDate not supplied

Stability of firefly luciferase in tricine buffer and in a commercial enzyme stabilizer.

A solution of firefly luciferase in AuthentiZyme Enzyme Stabilizer retains full activity when stored in an ice bath (0.5 degrees C) during one day. These solutions have the advantage that no additional protein (other than the luciferase) is present, which is desirable for proteolytic digestion and protein derivatization experiments. For longer-term experiments, firefly luciferase solutions in 0.05 mol/l Tricine buffer at pH 7.8, 10 mmol/l MgSO4, 1 mmol/l EDTA, and 1 mmol/l DTT which contain 100 micrograms/ml of bovine serum albumin are stable for 6 weeks if frozen and thawed only once.

Animals↗

PubMedDate not supplied

In memoriam Dr Marlene DeLuca. 1987 O. M. Smith Lecture. Firefly luciferase: mechanism of action, cloning and expression of the active enzyme.

The untimely death of Marlene DeLuca in 1987 has deprived the scientific community of an outstanding expert on bioluminescence. Earlier in that year she was honoured as the thirty-ninth recipient of the Otto Mitchell Smith Lectureship Award at Oklahoma State University, Stillwater, Oklahoma. On 20 March 1987 Dr DeLuca presented a scientific lecture entitled 'Firefly Luciferase--Mechanism of Action, Cloning, and Expression of the Active Enzyme' and a popular lecture at the banquet that evening entitled 'Light and Life'. She was selected for her excellence in research, her oral presentation ability, and her personableness. Marlene was the first woman so honoured. To honour Dr Otto M. Smith the Alpha Delta Chapter of Phi Lambda Upsilon, a national chemistry honorary organization, inaugurated The Otto Mitchell Smith Lectureship in 1948 at Oklahoma State University. Former awardees include Nobel Laureates H. C. Brown, Stanford Moore, and Arthur Kornberg and the following prominent biochemists/molecular biologists: Robert A. Alberty, University of Wisconsin; Daniel E. Koshland, Brookhaven National Laboratory; Sol Spiegelman, University of Illinois; Carl Djerassi, Stanford University; and John T. Edsall, Harvard University. The lectureship honours Dr O. M. Smith, who was Director of the Research Foundation, professor, and Head of the Departments of Chemistry and Chemical Engineering. As a tribute to Dr DeLuca's outstanding contribution to bioluminescence we reproduce here the edited text of her Otto Mitchell Smith Lectureship and a selected bibliography of her work on firefly bioluminescence.

Awards and Prizes↗

PubMedDate not supplied

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters.

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH-luc fusion to be observed by a dark-adapted eye and photographed.

Animals↗

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↗

PubMed1999

An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA).

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached. Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index.

Adenosine Deaminase↗

PubMed1999

Measurement of firefly luciferase reporter gene activity from cells and lysates using Escherichia coli arsenite and mercury sensors.

The structural gene encoding firefly luciferase from Photinus pyralis is a widely used reporter both in traditional monitoring of gene expression and in bacterial sensors. Its activity can be detected from living cells (in vivo) without disruption or from cell-free lysate (in vitro). We compared the two measurement methods by using an overall toxicity detecting strain Escherichia coli MC1061(pCSS810), a mercury-sensing strain E. coli MC1061(pTOO11), and two new arsenic sensor strains MC1061(pTOO31) and AW3110(pTOO31) which were constructed for this study. Plasmid pTOO31 was constructed by inserting the ars promoter and the arsR gene from plasmid R773 to control firefly luciferase gene expression. Both in vivo and in vitro methods correlated well with the strains tested [correlation coefficients R = 0.99484 and 0.99834] and gave highly comparable results with standard solutions of arsenite or mercury ions and from six environmental water samples spiked with the ions. Use of the in vivo method resulted in lower variation between replicates of the same sample (CVs ranging from 3.9 to 7.2%) and also between different samples (from 8.6 to 25.9%) compared to the in vitro method (CVs ranging from 8.6 to 17.8% for replicates and from 13.1 to 36.3% for different samples).

Animals↗

PubMed2000

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors.

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.

Animals↗

PubMed2002

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran.

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.

Adenosine Triphosphate↗

PubMed1999

Firefly luciferase as a reporter to study gene expression in Streptococcus mutans.

The utility of firefly luciferase as a reporter was tested in Streptococcus mutans. Under control of an endogenous promoter, the luciferase coding sequence was strongly expressed, while a promoterless version was indistinguishable from the background. Luciferase activity was easily extracted and the assay rapid and reproducible. In addition, the half-life of luciferase activity was found to be comparable to those of other frequently used reporters. Thus, firefly luciferase can readily be used as a reporter in S. mutans, a useful alternative to methods requiring radioactive isotopes.

Animals↗