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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↗

PubMed1998

Opiate analgesics' dual role in firefly luciferase activity.

The effects of three opiate analgesics, isolated from opium, on the firefly luciferase enzyme have been studied. Morphine (MN), 6-acetylmorphine (MAM), and diacetylmorphine (DAM) inhibited the enzyme activity at different levels. At lower concentrations, MN and MAM enhanced enzyme activity, effecting inhibition at higher concentrations. However, DAM inhibited the enzyme activity at all concentrations investigated. The stimulating activity of MN and MAM is attributed to the hydrophilic interaction of the proton donor-acceptor type with the polar regions of the luciferase located outside the binding pocket of the active site. The inhibition at higher concentrations of MN and MAM and at all concentrations of DAM is found to be competitive in nature, with the analgesics competing for the binding of the enzyme's natural substrate luciferin. The binding site of the luciferase could accommodate only one analgesic molecule. Binding constants determined from bioluminescence studies showed that the inhibitor binding site is hydrophobic in nature. The inhibition constants of analgesics are in the order MN > MAM > DAM. The greater binding of DAM to luciferase is attributed to its ability to form a ground state complex with ATP and greater hydrophobicity. At higher concentrations of ATP, the binding constants increased. The results obtained are explained assuming that the firefly luciferase acts as a subtype mu-opioid receptor model.

Adenosine Triphosphate↗

PubMed1999

Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.

Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.

Amino Acid Substitution↗

PubMed1985

ATP-dependent bioluminescence in the firefly squid, Watasenia scintillans.

The Japanese firefly squid, Watasenia scintillans, emits intense flashes of light from three tiny luminous organs that are located at the tip of each of a pair of ventral arms. Light is also produced from hundreds of other minute organs that are scattered over the body. The luminescence is due to an ATP-dependent reaction, with an optimal pH of 8.80. The decay of light intensity follows first-order kinetics and the decay constant is independent of initial ATP concentration. The light emission also requires MgCl(2), a soluble component, and an insoluble component that is membrane bound. Squids represent a major group of organisms unrelated to fireflies in which ATP is required for bioluminescence.

Journal Article↗

PubMed1990

An upstream region of the mouse ZP3 gene directs expression of firefly luciferase specifically to growing oocytes in transgenic mice.

The gene encoding the mouse egg primary receptor for sperm, a zona pellucida glycoprotein called ZP3, is expressed exclusively in growing oocytes within ovaries of sexually immature and mature female mice. We have constructed a transgene in which 6.5 kilobases of ZP3 gene 5'-flanking sequence is fused to the coding region of the firefly luciferase gene, and we have generated four independent lines of transgenic mice. In these animals, the transgene is expressed exclusively in ovaries. Furthermore, within ovaries, expression is confined to growing oocytes, and luciferase activity can be detected by assaying individual, isolated oocytes. The pattern of firefly luciferase expression during oocyte growth is similar to that observed in previous studies of ZP3 expression during oogenesis in mice. Observations reported here strongly suggest that cis-acting elements present in the ZP3 gene 5'-flanking region regulate oocyte-specific and, therefore, sex-specific expression of the sperm receptor gene during mouse development. They also suggest that such elements can be used to direct expression of cloned genes specifically to oocytes of transgenic mice and to evaluate the effects of such expression on various aspects of early mammalian development.

Animals↗

PubMed1991

Modulation of the general anesthetic sensitivity of a protein: a transition between two forms of firefly luciferase.

The activities of most proteins are relatively insensitive to general anesthetics. A notable exception is firefly luciferase, whose sensitivity to a wide range of anesthetic agents closely parallels that of whole animals. We have now found that this sensitivity can be controlled by ATP. The enzyme is insensitive at low (microM) concentrations of ATP and very sensitive at high (mM) concentrations. The differential sensitivity varies from anesthetic to anesthetic, being greatest (about a 100-fold difference) for molecules with large apolar segments. This suggests that anesthetic sensitivity is modulated by changes in the hydrophobicity of the anesthetic-binding pocket. Parallel changes in the binding of the substrate firefly luciferin, for which anesthetics compete, indicate that anesthetics bind at the same site as the luciferin substrate. These changes in the nature of the binding pocket modify not only the sensitivity to anesthetics but also the position of the "cutoff" in the homologous series of primary alcohol anesthetics; the cutoff position can vary from octanol to pentadecanol, depending upon the concentration of ATP. Our results suggest that particularly sensitive anesthetic target sites in the central nervous system may possess anesthetic-binding pockets whose polarities are regulated by neuromodulatory agents.

Adenosine Triphosphate↗

PubMed1995

Lanterns of the firefly Photinus pyralis contain abundant diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase activity.

The enzyme diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase has been purified to homogeneity from firefly lanterns. It is a single polypeptide of M(r) 16,000 with a Km for Ap4A of 1.9 microM and kcat = 3.6 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 7.5 nM) and non-competitively by fluoride ions (Ki = 50 microM). The specific activity of the enzyme in crude extracts of at least 20 milliunits/mg protein is 10-100 times higher than in any other eukaryote so far examined. Interestingly, firefly luciferase is known to synthesize Ap4A and related adenine-containing dinucleoside tetraphosphates in vitro. The high activity of Ap4A hydrolase in lanterns may be related to this ability and could be relevant to the use of the luciferase gene as a reporter gene.

Acid Anhydride Hydrolases↗

PubMed1997

Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.

Affinity Labels↗

PubMed1990

Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins.

We have previously shown that the peroxisomal targeting signal in firefly luciferase consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine (Gould, S.J., G.A. Keller, N. Hosken, J. Wilkinson, and S. Subramani, 1989. J. Cell Biol. 108:1657-1664). Antibodies were raised against a synthetic peptide that contained this tripeptide at its COOH terminus. Immunofluorescence and immunocryoelectron microscopy revealed that the anti-peptide antibodies specifically detected peroxisomes in mammalian cells. Further characterization revealed that the antibodies were primarily directed against the COOH-terminal three amino acids of the peptide. In Western blot experiments, the antibodies recognized 15-20 rat liver peroxisomal proteins, but reacted with only a few proteins from other subcellular compartments. These results provide independent immunological evidence that the peroxisomal targeting signal identified in firefly luciferase is present in many peroxisomal proteins.

Amino Acid Sequence↗

PubMed1990

Mycoplasma somnilux sp. nov., Mycoplasma luminosum sp. nov., and Mycoplasma lucivorax sp. nov., new sterol-requiring mollicutes from firefly beetles (Coleoptera: Lampyridae).

Strain PYAN-1T (T = type strain), which was isolated from a pupal gut of the firefly beetle Pyractonema angulata, and strains PIMN-1T and PIPN-2T, which were isolated from guts of adult Photinus marginalis and Photinus pyralis fireflies, respectively, were demonstrated to be sterol-requiring mollicutes. Cells of the three strains were shown by electron and dark-field microscopy to be small, pleomorphic, nonhelical, nonmotile bodies surrounded by single membranes. No evidence of a cell wall was observed, and the organisms were not susceptible to 500 U of penicillin per ml. The three strains grew rapidly in SP-4 broth medium. Strains PIMN-1T and PIPN-2T grew in medium supplemented with bovine serum fraction, but strain PYAN-1T did not. All three strains grew on solid media when the cultures were incubated aerobically, but only strains PYAN-1T and PIPN-2T formed colonies when anaerobic conditions were employed. The three strains catabolized glucose but hydrolyzed neither arginine nor urea. All of the strains grew at temperatures of 18 to 32 degrees C; strains PYAN-1T and PIMN-1T also grew at 10 degrees C. The optimal temperature for growth for strains PYAN-1T and PIPN-2T was 30 degrees C; strain PIMN-1T grew equally well at 30 or 32 degrees C. None of the three strains grew at 37 degrees C. The genome sizes of strains PYAN-1T, PIMN-1T, and PIPN-2T were about 527 (478 to 589), 570 (480 to 630), and 762 (635 to 871) megadaltons, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A novel circadian phenotype based on firefly luciferase expression in transgenic plants.

A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high-amplitude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in individual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock.

Animals↗

PubMed1996

Crystallization and preliminary diffraction studies of firefly luciferase from Photinus pyralis.

Firefly luciferase is a 62 kDa molecular weight enzyme which catalyzes a light-emitting reaction. Crystals of Photinus pyralis luciferase have been obtained by the microbatch technique, using polyethylene glycol as a precipitating agent. Firefly luciferase crystallizes as long needles which belong to the tetragonal space group P4(1)2(1)2, with cell dimensions a = 119.5, b = 119.5, c = 95.4 A. One molecule is present in the asymmetric unit. Diffracted intensities beyond 2.0 A resolution have been measured from frozen crystals using synchrotron radiation.

Journal Article↗

PubMed1991

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.

Animals↗

PubMed1995

Cloning, expression and sequence analysis of cDNA for the luciferases from the Japanese fireflies, Pyrocoelia miyako and Hotaria parvula.

Cloning and sequence analysis of cDNA for the luciferases of Pyrocoelia miyako and Hotaria parvula were carried out (GenBank accession numbers L39928 and L39929, respectively). The amino acid sequence, deduced from the nucleotide sequence, showed P. miyako luciferase to consist of 548 amino acid residues with a molecular weight of 60,955, while the luciferase of H. parvula consisted of 548 amino acid residues with a molecular weight of 60,364. Pyrocoelia miyako luciferase showed 82.1% homology with the luciferase of Photinus pyralis and less than 70% homology with other firefly luciferases, whereas H. parvula luciferase showed 98%, 82.5% and 81.2% homology with the luciferases of Luciola mingrelica, Luciola lateralis and Luciola, cruciata respectively. Two regions in the enzymes were found to be highly conserved. The amino acid sequences were used to construct a phylogenetic tree, which showed that the fireflies could be divided into two groups.

Amino Acid Sequence↗

PubMed1979

Octopamine receptors, adenosine 3',5'-monophosphate, and neural control of firefly flashing.

An adenylate cyclase activated as much as 25-fold by low concentrations of octopamine has been identified in the firefly lantern. The relative potency of octopamine and various other amines in stimulating this enzyme, and effects of antagonists in blocking octopamine activation, correlate well with the known effects of these agents in affecting light production. In addition to suggesting a role for adenosine 3',5'-monophosphate (or pyrophosphate) in the neural control of firefly flashing, identification of this potent enzyme should facilitate the characterization of phenylethylamine receptors in excitable tissue.

Adenylyl Cyclases↗

PubMed1993

Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring.

A system for stable tagging of gram-negative bacteria with the firefly luciferase gene, luc, is described. A previously constructed fusion constitutively expressing luc from the lambdap(R) promoter was used. Stable integration into the bacterial genome was achieved by use of mini-Tn5 delivery vectors. The procedure developed was applied for tagging of representative gram-negative bacteria, such as Escherichia coli, Rhizobium meliloti, Pseudomonas putida, and Agrobacterium tumefaciens. The system permitted the detection of tagged R. meliloti in the presence of more than 10 CFU per plate without the use of any selective markers (such as antibiotic resistance genes). No significant differences in growth rates or soil survival were found between the marked strain and the wild-type strain. Studies of bioluminescent R. meliloti also revealed a good correlation between cell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive method for the detection and enumeration of bacteria in the environment.

Journal Article↗

PubMed1983

Firefly luciferase ATP assay as a screening method for bacteriuria.

A rapid (15 min) test for bacteriuria based on firefly luciferase analysis of bacterial ATP has been evaluated in 2,018 clinical urine specimens. The test procedure involves removal of nonbacterial ATP by treatment of urine with Triton X-100 and apyrase, extraction of bacterial ATP by boiling, and bioluminescent analysis of bacterial ATP by firefly luciferase, using a luminometer. For comparison, the widely used nitrite test was included in the study as an example of an alternative rapid chemical test. The test was set up to distinguish between specimens yielding greater than 10(5) CFU/ml and specimens yielding less than 10(5) CFU/ml. A level of 13.5 nM ATP was chosen to define the limit between negative and positive results. At this discriminatory level, 92% of specimens yielding greater than 10(5) CFU/ml and 88% of specimens yielding less than 10(5) CFU/ml were correctly classified with the luciferase method, whereas corresponding figures for the nitrite test were 55 and 99%, respectively. Of the 12% false luciferase positives, 20% were shown to contain greater than 10(5) CFU/ml on prolonged incubation, thus reducing the false-positive rate to 10%. Of the 8% false luciferase negatives, 65% had low levels of CFU in the range of 10(5) to 10(6).

Adenosine Triphosphate↗

PubMed1999

Development of a firefly luciferase-based assay for determining antimicrobial susceptibility of Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis (Johne's disease) is a fatal disease of ruminants for which no effective treatment is available. Presently, no drugs against Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, are approved for use in livestock. Additionally, M. paratuberculosis has been linked to a human chronic granulomatous ileitis (Crohn's disease). To assist in the evaluation of antimicrobial agents with potential activity against M. paratuberculosis, we have developed a firefly luciferase-based assay for the determination of drug susceptibilities. The microorganism used was M. paratuberculosis K-10(pYUB180), a clinical isolate carrying a plasmid with the firefly luciferase gene. The MICs determined by the broth macrodilution method were as follows: amikacin, 2 microg/ml; Bay y 3118, 0.015 microg/ml; clarithromycin, 1.25 microg/ml; D-cycloserine, 25 microg/ml; ethambutol, 20 microg/ml; and rifabutin, 0.5 microg/ml. The strain was resistant to isoniazid and kanamycin. The results obtained by the luciferase assay were identical or fell within 1 doubling dilution. These results suggest that a combination of amikacin, clarithromycin, and rifabutin may be the most efficacious therapy for the treatment of M. paratuberculosis infections and that the use of fluoroquinolone class of antibiotics deserves further consideration. We demonstrate that the luciferase drug susceptibility assay is reliable for M. paratuberculosis and gives results within 7 days, whereas the broth macrodilution method requires 14 days.

Anti-Bacterial Agents↗