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At least 19 recordsLinked to original sources
PubMed1976

Studies on luciferase from Photobacterium phosphoreum. VIII. FMN-H2O2 initiated bioluminescence and the thermodynamics of the elementary steps of the luciferase reaction.

Bacterial luciferase from Photobacterium phosohoreum was found to produce bioluminescence on reaction with FMN and H2O2 in the presence of aldehyde. This luminescence is presumably produced by the same X1 intermediate as that found in FMNH2-O2 initiated luminescence. From the ratio of the light intensities of the FMN-H2O2 initiated reactions, we calculated the association constant of the reaction, luciferase+FMN+H2O2in equilibriumX1, and estimated its temperature dependence. From these results we calculated the thermodynamic parameters of the reaction, luciferase+FMNH2+O2in equilibriumX1. We found that the free energy level of X1 is only 3.2 kcal below that of fr-e FMN and H2O2. We also estimated the thermodynamic parameters of other steps of the luminescent reaction. The values obtained showed that the formation of X1 from luciferase, FMNH2 and O2 involves a positive entropy change, but the intermediate is in a state stabilized against decomposition. Results also suggest a considerable degree of electronic rearrangement on formation of the excited-state molecule from the X1-aldehyde complex.

Aldehydes↗
PubMed1978

Studies on luciferase from Photobacterium phosphoreum. XI. Interaction of 8-substituted FMNH2 with luciferase.

The interaction of bacterial luciferase from Photobacterium phosphoreum with reduced flavin was investigated using various 8-substituted FMNH2 analogs. Flavins tested were FMNH2 and FMNH2 substituted at the 8 position with HO-, CH3O-, C2H5O-, Cl-, Br-, I-, H2N-, (CH3)HN-, and (ch3)2n. 8-ch30-, c2h5o-, cl-, and Br-FMNH2 showed luminescent activity in the luciferase reaction with emission peaks at various wavelengths. 8-HO- and I-FMNH2 were competitive inhibitors toward FMNH2 in the luminescent reaction. 8-Amino analogs of FMNH2 showed no luminescent or inhibitor activity. The dissociation constant of the luciferase-FMNH2 analog complex was determined kinetically as a substrate or inhibitor constant. A contribution of the imino group at position 5 in the isoalloxazine ring to the FMNH2 binding to luciferase was suggested by a Hammett plot of the dissociation constants.

Flavin Mononucleotide↗
PubMed1975

Photoexcited bacterial bioluminescence. Identity and properties of the photoexcitable luciferase.

Properties of photoexcitable luciferase are compared with those of luciferase, both isolated from the bacterium Beneckea harveyi. The proteins have the same molecular weight, are similarly charged at pH 8, and can be inactivated, with comparable efficiencies, by antibodies against either pure luciferase (a heterodimeric protein) or individual subunits thereof. Compared with luciferase, photoexcitable luciferase has a broader pH range for optimal activity, is more stable under acidic conditions, is less stable under alkaline conditions, and is more resistant at neutral pH to inactivation by heat, urea, and trypsin; A flavine-like chromophore, designated B, can be isolated from photoexcitable luciferase. The binding of B to luciferase restores all the properties characteristic of photoexcitable luciferase. Moreover, photoexcitable luciferases from mutants selected to have heat labile luciferases are also thermally unstable. It is concluded that photoexcitable luciferase actually consists of a luciferase-B complex which is conformationally distinct from luciferase under certain conditions.

Animals↗
PubMed1992

Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae.

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.

Animals↗
PubMed1978

Structural studies on bacterial luciferase using energy transfer and emission anisotropy.

The distance between specific sites on bacterial luciferase was estimated by energy transfer. Luciferase was fluorescently labeled by reaction of an essential sulfhydryl group with N-(1-pyrene)maleimide and N-[p-(2-benzoxazolyl)phenyl]meleimide. Both of the modified enzymes bind 8-anilino-1-naphthalenesulfonate (Ans) with affinities similar to that exhibited by the native luciferase. Using each of the two fluorescent probes as a donor and the bound Ans as an acceptor, the energy transfer efficiencies were determined by the resulting enhancement of fluorescence of the acceptor. The corresponding distance was calculated to be in the range of 21 to 37 A. Energy-transfer studies were also carried out using fluorescence lifetime measurements of bound ANS, acting as a donor with bound FMN as an acceptor. The corresponding distance was calculated to be between 30 and 58 A. Using samples of luciferase:Ans complex and luciferase modified with N-(1-pyrene)maleimide, the rotational correlation time of the enzyme-dye conjugate as awhole was found to be 47 +/- 2 ns. The observed rotational correlation time is much longer than that calculated for luciferase assuming a spherical structure, thus indicating an elongated form for the luciferase-dye conjugate.

Anilino Naphthalenesulfonates↗
PubMed1975

Photoexcited bacterial luminescence. Spectral properties and mechanistic implication of a reduced flavine-like prosthetic group associated with photoexcitable luciferase.

A prosthetic group, designated B, has been isolated from bacterial photoexcitable luciferase and found to possess spectral and photochemical properties characteristic of substituted reduced flavines. Its fluorescence when bound to luciferase has an excitation maximum at 375 nm, correlating well with the absorption spectrum, and an emission peaking at 495 nm. However, free B is nonfluorescent in aqueous solution at ambient temperature. Both free and luciferase-bound B show similar negative circular dichroism in the region 330-475 nm with troughs at 375 and 380 nm, respectively. In the luciferase reaction initiated by FMNH2, B is an inhibitor competitive with FMNH2. Irradiation of photoexcitable luciferase converts B to FMN, the latter identified spectrally, enzymatically, and chromatographically. These findings lead to the suggestion that B is a substituted FMNH2. The luciferase-bound B resembles but is not identical with the normal flavine intermediate obtainable by reacting luciferase with reduced flavine mononucleotide and oxygen. It is hypothesized that B is a false intermediate of the bacterial bioluminescence reaction, and a mechanism for the photoexcited bioluminescence reaction is suggested.

Binding Sites↗
PubMed1975

Differential effects of 8-anilino-1-naphthalenesulfonate upon binding of oxidized and reduced flavines by bacterial luciferase.

Upon binding to bacterial luciferase, both the absorption and the fluorescence excitatiom maxima of 8-anilino-1-naphthalensulfonate (ANS) shift from 353 to 370 nm while the fluorescence emission optimum shifts from 540 to 480 nm, and the fluorescence quantum yield increases from 0.003 to 0.39, indicating that the environment of the ANS binding site is hydrophobic. ANS binds to luciferase with dissociation constants of 1.9 X 10(-5) and 2.3 X 10(-5) M at 5 and 23 degrees, repsectively. As with both oxidized flavine mononucleotide (FMN) and reduced flavine mononucleotide (FMNH2), ANS also binds to luciferase with a stoichiometry of 1 site per dimeric luciferase molecule. ANS acts as a luciferase inhibitor, competitive with FMNH2, with an inhibitor constant of 2.3 X 10(-5) M at 23 degrees. However, the binding of ANS does not significantly displace FMN from binding to luciferase. Interactions of FMN and FMNH2 with luciferase are thus differentially regulated by the ANS binding.

Anilino Naphthalenesulfonates↗
PubMed1975

Bacterial luciferase. Binding of oxidized flavin mononucleotide.

Bacterial luciferase catalyzes a bioluminescent oxidation of reduced flavin mononucleotide; the products include a photon and oxidized FMN. The experiments reported here show that luciferase binds oxidized flavin mononucleotide in a 1:1 molar ratio with an apparent dissociation constant of 1.2 times 10-4 M at 3 degrees in 0.05 M 2,2-bis(hydroxymethyl)-2,2'2"-nitriloethanol (bis-tris), pH 7.0. Analysis of the binding at temperatures between 3 and 30 degrees indicates an enthalpy of binding (delta H a) of minus 10.0 kcal per mol. The absorption spectrum of luciferase-bound FMN shows considerable alteration relative to that of free flavin. There is one major peak at 366 nm, and the 445-nm band is resolved into two distinct peaks at 434 and 458 nm; this spectrum is indicative of binding in a nonpolar environment. The circular dichroism spectrum of FMN bound to luciferase has structure which correlates well with the optical absorption spectrum of the bound flavin. The detail in the spectra of the bound FMN probably reflects the resolution of vibrational structure which is blurred in polar environments. The optical activity shown by the CD spectrum presumably results from binding in an electronically asymmetric fashion. Although FMN free in solution is highly fluorescent, FMN bound to luciferase is nonfluorescent, thus indicating that the emitting species is not an excited state of product FMN located in the same site in which luciferase binds oxidized FMN.

Calorimetry↗
PubMed1977

Bacterial luciferase subunits are synthesized in equal quantities.

Synthesis of luciferase, an alpha beta dimer, occurs during a relatively short period of time near the end of exponential growth of Beneckea harveyi. The rates of synthesis of the individual alpha and beta chains of luciferase are compared by quantitating the molar ratio of total cellular alpha to that of beta at different growth stages. The addition of exogenous alpha or beta subunit to crude lysates of cells taken prior to and during the luciferase induction produces no increase in luciferase activity. This result, together with previous evidence that there are no antigenically cross-reacting precursors, allows us to conclude that luciferase alpha and beta chains are synthesized pari passu, i.e. in equal proportions, and exist primarily in the alpha beta dimeric form. It is also shown that insoluble sedimentable cellular materials contain no detectable luciferase subunits.

Gram-Negative Anaerobic Bacteria↗
PubMed2025

Luciferase-Based Reporter Assay for the Assessment of Aurora A-Kinase Activity in Mitotic Cycle.

Luciferase-based reporter assay is an important tool that employs bioluminescence to quickly and precisely investigate the gene of interest's promoter activity by reporter gene expression at the transcriptional level. The promoter of the gene of interest is fused with the reporter gene (a gene that produces luciferase enzymes) and then transfected into the cells. Luciferase is an enzyme that catalyzes a chemical reaction to produce light. The bioluminescence activity of the luciferase gene in the transfected cells is directly proportional to the expression of the gene of interest, which is measured by using a luminometer. In this chapter, we outline the use of a dual-reporter luciferase assay to measure Aurora A kinase activity during the mitotic cycle.

Genes, Reporter↗
PubMed1979

Isolation and properties of bacterial luciferase-oxygenated flavin intermediate complexed with long-chain alcohols.

Nonsubstrate long-chain aliphatic alcohols, carboxylic acids, and their methyl esters were found to complex reversibly with and stabilize an oxygenated flavin-luciferase intermediate, with alcohols being more effective in stabilizing the intermediate. Dissociation constants for the binding of alcohols to luciferase intermediate are in the order of K8 greater than K10 greater than K12 congruent to K14 where the subscripts represent the numbers of carbon atoms of various alcohols. Thermodynamic activation parameters for the decay of oxygenated flavin-luciferase intermediate complexed with alcohols or aldehydes were determined, and similarities were noted between alcohol and aldehyde complexes. Luciferase intermediate complexes formed with 1-decanol and 1-tetradecanol were isolated at 0 degrees C in neutral phosphate buffer, and both showed absorption properties characteristic of 4a-substituted dihydroflavins. The 1-tetradecanol-intermediate species contained one favin per luciferase molecule. Initially this complex was weakly fluorescent, but upon exposure to 370-nm light it was transformed to a highly fluorescent species. The latter shows a fluorescence excitation peak at 370 nm, and its fluorescence emission (lambda max 505 nm) and quantum yield (0.17) closely correspond to that of bioluminescence in vitro. Both the weakly and the highly fluorescent species exhibit full bioluminescence activities when reacted with decanal.

Alcohols↗
PubMed1976

Chemical modification of bacterial luciferase with ethoxyformic anhydride: evidence for an essential histidyl residue.

Bacterial luciferase is a heteropolymeric protein (alphabeta) that catalyses the conversion of chemical energy to light by oxidation of a reduced flavin mononucleotide and a long chain aliphatic aldehyde. Elucidation of the specific amino acid residues involved in the enzymatic reaction is essential for understanding the mechanisms of the bioluminescent reaction. Luciferase has been found to be inactivated by ethoxyformic anhydride with a second-order rate constant of 146 M-1 min-1 at pH 6.1 and 0 degrees C with a concomitant increase in absorbance at 240 nm due to formation of ethoxyformylhistidyl derivatives. Activity could be restored by hydroxylamine and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Both substrates, FMNH2 and aldehyde, protected the enzyme against inactivation, suggesting that the modification occurred at or near the active site. Incorporation of [14C]ethoxyformyl groups in luciferase indicated that inactivation resulted from the modification of about three histidyl residues, one histidine being found on the alpha subunit and two on the beta subunit. Hybridization experiments, in which ethoxyformylluciferase, alphambetam, was complemented with native subunits, alpha or beta, showed that the hybrid alphambetam, has the same activity as alphambetam whereas the activity of the hybrid alphabetam, was close to that of the reconstituted luciferase alphabeta. The results indicate that modification of only one histidyl residue on the alpha subunit inactivates luciferase and suggest that this histidyl residue plays an essential role in the mechanism of the bacterial bioluminescent reaction.

Aldehydes↗
PubMedDate not supplied

Firefly luciferase gene transmission and expression in transgenic medaka (Oryzias latipes).

Plasmids containing the luciferase gene from the firefly (Photinus pyralis) fused to the Chinese hamster metallothioneine I promoter (ChMTI) were microinjected into the pronuclei of medaka (Oryzias latipes) eggs, which were then artificially inseminated. Evidence of integration into the genome was gained from observation of germ-line transmission in a mendelian fashion from the F1 to the F2 generation. However, gene expression (light emission) could not be demonstrated in the established transgenic line. In a separate program, transient expression of gene constructs containing the luciferase gene fused to various promoters was compared in medaka embryos. Plasmids were microinjected into pronuclei, and homogenates from 3-day-old embryos were measured for light emission using a luminometer. Among the various promoters tested (SV40, RSV-LTR, ChMTI, HSP70, and mouse albumin), the highest levels of luciferase gene expression were observed in gene constructs containing ChMTI and HSP70 gene promoters. Expression in these two constructs was significantly increased following administration of ZnSO4 or heat treatment, respectively. Plasmids were also introduced into goldfish fibroblast-like cells in vitro, in which enzymatically active luciferase was transiently expressed. Assaying for expression of luciferase provided a rapid and sensitive method for monitoring promoter activity. The potential usefulness of this fish species for cancer research is discussed based on accumulated information from carcinogenesis studies.

Animals↗
PubMed2025

Functional Characterization of Luciferase in a Brittle Star Indicates Parallel Evolution Influenced by Genomic Availability of Haloalkane Dehalogenase.

Determining why convergent traits use distinct versus shared genetic components is crucial for understanding how evolutionary processes generate and sustain biodiversity. However, the factors dictating the genetic underpinnings of convergent traits remain incompletely understood. Here, we use heterologous protein expression, biochemical assays, and phylogenetic analyses to confirm the origin of a luciferase gene from haloalkane dehalogenases in the brittle star Amphiura filiformis. Through database searches and gene tree analyses, we also show a complex pattern of the presence and absence of haloalkane dehalogenases across organismal genomes. These results first confirm parallel evolution across a vast phylogenetic distance, because octocorals like Renilla also use luciferase derived from haloalkane dehalogenases. This parallel evolution is surprising, even though previously hypothesized, because many organisms that also use coelenterazine as the bioluminescence substrate evolved completely distinct luciferases. The inability to detect haloalkane dehalogenases in the genomes of several bioluminescent groups suggests that the distribution of this gene family influences its recruitment as a luciferase. Together, our findings highlight how biochemical function and genomic availability help determine whether distinct or shared genetic components are used during the convergent evolution of traits like bioluminescence.

Echinodermata↗
PubMed2026

Screening of Estrogenic and Antiestrogenic Effects of Estradiol, Bisphenol A, and Fulvestrant Using 2D and 3D Breast Cancer Cell Systems With a Luciferase Reporter Gene Assay.

Endocrine-disrupting chemicals (EDCs) like bisphenol A (BPA) pose health risks by interfering with hormones. This study develops and utilizes in vitro 2D and 3D cell models to evaluate the estrogenic and antiestrogenic properties of compounds. Human breast cancer cell lines T47D and MCF7, stably transfected with a luciferase reporter gene (ERE-LUC), were first compared in 2D. Due to the significantly higher sensitivity and responsiveness observed in the T47D line during preliminary 2D screenings, this cell line was exclusively selected for the development of the 3D spheroid model. Cells were treated with 17β-estradiol (E2), BPA, and Fulvestrant (FUL) to assess cell viability and luciferase activity. In 2D models, T47D ERE-LUC cells showed higher responsiveness than MCF7 ERE-LUC, which failed to show significant luciferase induction with E2. In the 3D T47D model, cells exhibited significant and robust changes in luciferase activity in response to E2 and BPA, highlighting the enhanced fidelity of 3D cultures in replicating tissue conditions compared to their 2D counterparts. The study highlights the effectiveness of 3D models over 2D in evaluating estrogenic activity. Specifically, the 3D T47D ERE-LUC system serves as a superior, sensitive, and reliable platform for screening EDCs, offering benefits in cost, data speed, and reduced in vivo reliance.

Humans↗
PubMed1977

Control of luciferase synthesis in a newly isolated strain of Photobacterium leiognathi.

In previous studies with luminous bacteria of all different species it has been reported that the synthesis of luciferase is autoinducible: during growth at low cell densities synthesis is effectively repressed while after induction, at higher cell densities, the rate of synthesis of enzyme is up to five times the growth rate. In this paper we report on newly isolated strains of Photobacterium leiognathi which show continued luciferase synthesis irrespective of the cell density. The specific synthesis rate may nevertheless differ from the rate of growth and depends on the luciferase content of the inoculated cells. A ratio of 1 was established for cells having a maximum luciferase content varying to a ratio of about 2 for cells that contained only 1% of the maximum.

Luciferases↗
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↗