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At least 19 recordsLinked to original sources
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↗
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↗
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↗
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

Purification and properties of Renilla reniformis luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.

Amino Acids↗
PubMed1977

Substrate and substrate analogue binding properties of Renilla luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis catalyzes the oxidative decarboxylation of luciferin consuming 1 mol of O2 per mol of luciferin oxidized and producing 1 mol of CO2, 1 mol of oxyluciferin, and light (lambdaB, 480 nm) with a 5.5% quantum yield. In this work we have examined the binding characteristics of luciferin, luciferin analogues, and competitive inhibitors of the luciferin-luciferase reaction. The results show that luciferin binding and orientation in the single luciferin binding site of luciferase are highly specific for and dependent upon the three group substituents of the luciferin molecule while the imidazolone-pyrazine nucleus of luciferin is not directly involved in binding. Anaerobic luciferin binding promotes a rapid concentration-dependent aggregation of luciferase which results in irreversible inactivation of the enzyme. This aggregation phenomenon is not observed upon binding of oxyluciferin, luciferyl sulfate, or luciferin analogues in which the substituent at the 2 position of the imidazolone-pyrazine ring has been substantially altered.

Animals↗
PubMed1979

Covalent structure of subunits of bacterial luciferase: NH2-terminal sequence demonstrates subunit homology.

The heterodimeric subunit structure of bacterial luciferase was demonstrated more than 10 years ago. The enzymes from both Beneckea harveyi and Photobacterium fischeri have since been studied in detail; they each consist of two nonidentical subunits, designated alpha and beta. Both are required for bioluminescence activity, with the active center apparently confined to the alpha subunit. Amino acid sequence analysis of the NH2 termini of the alpha and beta subunits of the B. harveyi and P. fischeri luciferases not only confirms the earlier observation that the alpha subunits are homologous but also demonstrates that the NH2-terminal sequences of the beta subunits of the luciferases from the two genera are homologous. Furthermore, within each luciferase, the NH2-terminal sequences of the alpha and beta subunits are similar, suggesting the possibility that the genes coding, for alpha and beta may have arisen by gene duplication, presumably prior to divergence of the lines leading to present-day luminous bacteria.

Amino Acid Sequence↗
PubMed1975

Characterization and properties of Pholas luciferase as a metalloglycoprotein.

The luciferase of the bioluminescent boring mollusc, Pholas dactylus, has been purified by a new method which includes centrifugation in cesium chloride gradients. Homogeneous preparations have been obtained and molecular weight determinations and subunit analysis support the idea that this preparation is an oxyluciferin-luciferase complex. The preparation catalyzes oxidation of ascorbic acid in presence of H2O2, and this peroxidase activitity has been used for characterization (thermal and pH stabilities, activity as a function of pH, isoelectric point, turnover number). The existence of two atoms of copper has been established and their involvement in the peroxidase activity indicated. Chemical analyses have shown that Pholad luciferase is a glycoprotein and the existence of glucosamine, fucose, mannose, and galactose residues has been demonstrated. The apparent buoyant dentisty (1.340), the sedimentation coefficient (10.7 S), the Stoke's radius (83 A), the partial specific volum (0.707), and the molecular weight (350,000) have been determined. The frictional ratio (flf0 = 1.8) derived from the Stoke's radius indicates that the molecule is asymmetric. The quaternary structure has been examined. Subunits of molecular weight 150,000 and 46,000 have been observed. The latter has electrophoretic properties identical with luciferin or oxyluciferin.

Amino Acids↗
PubMed1979

[Kinetic properties of highly purified luciferase from fireflies Luciola mingrelica].

Luciferase of the fireflies Luciola mingrelica was isolated from dried lanterns of fireflies and purified by chromatography on DEAE-Sephadex. The homogeneity of the preparation was determined by polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme equal to 45000 was determined by disc electrophoresis in the presence of sodium dodecyl sulfate. The kinetic properties of the enzyme (V and Km for luciferin and ATP) within the pH-range of 7,0--8,5 were studied. The kinetic curves of the pH-dependences of log V and log Km for both substrates are bell-shaped, with a slope equal to 2. At pH optimum (7,7--7,9) the Km values for luciferin and ATP are 6,6 mkM and 0,3 mM, respectively. The properties of luciferase L. m. were compared to those of luciferase from fireflies Phophinus pyralis previously described in literature.

Animals↗
PubMed2026

Development of receptor and oncogene-responsive luciferase reporter vectors that are activated via kinase signaling to AP-1.

This study aimed to generate lentiviral vectors carrying an array of AP-1 motifs driving luciferase gene expression as reporters of mitogen-activated protein kinase (MAPK) activity. We created a series of vectors based on LeGO-iG that were used to generate stably transduced leukemia cell lines. A vector termed LEGO-AP1×6-GM55 containing an array of 6 AP-1 sites linked to the minimal CSF2 promoter was sufficient to support high levels of MAPK-inducible luciferase activity in leukemic cell lines that was suppressed by MAPK inhibitors. The inclusion of a putative chromatin priming element encompassing RUNX and ETS motifs increased the activity of these vectors. The additional inclusion of the full-length mouse CSF2 promoter, or the human DUSP5 promoter further increased the MAPK-dependent activity of these vectors in leukemic cells. These vectors support moderate levels of constitutive activity in cells carrying mutations that activate the RAS/RAF/MEK MAPK signaling pathway, and high-level activity after direct activation of MAPK signaling. They also respond to T-cell receptor activation via MAPK and Ca2+ signaling pathways. This resource will now make it easier to track receptor or oncogene-inducible MAPK activity in cultured cells, and potentially in tumors, close to real-time.

Humans↗
PubMed1979

Interactions of long-chain aldehydes with luciferase. A carbon-13 nuclear magnetic resonance study.

The interaction of long-chain aldehydes with bacterial luciferase has been studied by 13C NMR spectroscopy of natural-abundance and 13C-enriched 1-dodecanal. At high substrate/enzyme ratios, the spin-spin relaxation rates of C(1)-C(3) are faster than for the other carbons and are in the order C(1) greater than C(2) greater than C(3). The aldehyde is strongly bound in the active site along the entire length of the alkyl chain with the strongest interaction at the CHO group. At low substrate/enzyme ratios, interactions are apparent at C(10), which are removed upon denaturation of the enzyme. Spin-spin and spin-lattice relaxation rates were measured for odd-carbon 13C-enriched 1-dodecanal in the presence of luciferase. From the ratios of T1/T2 a single value of (1.8 +/- 0.7) X 10(-8) s was calculated for the rotational correlation time tc for the complex.

Aldehydes↗
PubMed1976

Effect of various ions on ATP determinations using the "luciferine-luciferase" system.

Various salts and buffers used in routine as part of the ATP extraction procedues induce an important inhibition of the peak light emission produced by the "luciferine-luciferase" system. The nature of the anion is more important in determining the inhibitory effect than the nature of the cation. The series obtained when placing the anions studied by order of increasing effectiveness is as follows Ac- less than Cl- less than I- less than ClO4-. KClO4 appears thus as a strong inhibitor of the enzyme activity. It appears moreover to act competitively with respect to ATP, one mole of inhibitor binding per mole of ATP active site. These results are discussed in connection with the use of the "luciferine-luciferase" system for ATP and other energy-rich compounds' concentration measurements.

Adenosine Triphosphate↗
PubMed2025

Use of Rift Valley Fever Virus Expressing NanoLuc Luciferase for the Assessment of Neutralizing Antibodies and Antivirals.

Rift Valley fever (RVF) is an arboviral zoonotic disease affecting many African countries with the potential to spread to other geographical areas. In this chapter we describe the use of a replication-competent recombinant (r)RVFV expressing NanoLuc Luciferase (Nluc) for in vitro studies. The determination of parameters such as neutralizing antibodies in serum samples, or the antiviral activity of drugs is usually carried out using standard assays based on the assessment of cytopathic effect on cell cultures. The use of a virus encoding a traceable reporter protein allows to correlate the presence or absence of infection with the detection of the product in the infected cultures, thus tracking the level of RVFV infection in an objective, quantitative manner. In addition to this quantitative measurement of results, our protocol offers two other advantages, such as a shorter time to read, given that 48 h post-infection the production of the reporter protein is enough to give an accurate result, and the use of an attenuated virus, which reduces the risk of exposure.

Rift Valley fever virus↗
PubMed1979

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 micrograms of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP in equilibrium with AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay.

Adenylate Kinase↗
PubMed1978

Activity and stability of the luciferase--flavin intermediate.

A luciferase intermediate in the bacterial bioluminescence system, which is formed by reaction of enzyme with reduced flavin mononucleotide (FMNH2) and oxygen, is shown to emit light with added aldehyde under anaerobic conditions. The reaction with oxygen is thus effectively irreversible under the conditions used. The flavin chromophore has an absorption maximum at about 370 nm and the potential activity (bioluminescence yield) in the further reaction of the isolated intermediate with aldehyde is strictly proportional to the amount of this flavin chromophore.

Anaerobiosis↗