A cysteine-free firefly luciferase retains luminescence activity

A mutant of Photinus pyralis luciferase in which all four native cysteine residues are converted to serines retains about 10% of wild-type activity. This mutant should prove useful as a starting point for the introduction of biophysical probes of conformational changes associated with enzyme function. The activities of the cysteine-free mutant and others in which two or three cysteines are converted to serines suggest, however, that small chemical changes can have substantial and interdependent effects on bioluminescence. The introduction of probes should therefore be approached cautiously.     

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.     

Effect of solvents on the catalytic activity of firefly luciferase

Various solvents stimulate the catalytic activity of firefly luciferase, up to sevenfold. Polyvinylpyrrolidone, polyethylene glycols, and nonionic detergents such as Triton X-100 were the most effective stimulators of the enzyme. Both peak light and total light emission were enhanced in the presence of these solvents indicating an increased turnover of the enzyme. The primary effect of the solvents is on the oxidative reaction rather than the activation reaction. All the experimental data support the hypothesis that the presence of solvent promotes the dissociation of the inhibitory product from the enzyme.     

Effect of ATP concentration and temperature on firefly luciferase activity

The dependence of luciferase activity in the homogenate of leaves of transgenic tobacco plants with chimeric firefly luciferase gene on ATP concentration and temperature was studied. The optimum ATP concentration was between 0.625 mM and 2.5 mM. The activity rapidly decreased if the homogenate was kept in 25°C and is completely lost during 30 min.     

Gaussia luciferase for bioluminescence tumor monitoring in comparison with firefly luciferase

Gaussia luciferase (Gluc) is a secreted reporter, and its expression in living animals can be assessed by in vivo bioluminescence imaging (BLI) or blood assays. We characterized Gluc as an in vivo reporter in comparison with firefly luciferase (Fluc). Mice were inoculated subcutaneously with tumor cells expressing both Fluc and Gluc and underwent Fluc BLI, Gluc BLI, blood assays of Gluc activity, and caliper measurement. In Gluc BLI, the signal from the tumor peaked immediately and then decreased rapidly. In the longitudinal monitoring, all measures indicated an increase in tumor burden early after cell inoculation. However, the increase reached plateaus in Gluc BLI and Fluc BLI despite a continuous increase in the caliper measurement and Gluc blood assay. Significant correlations were found between the measures, and the correlation between the blood signal and caliper volume was especially high. Gluc allows tumor monitoring in mice and should be applicable to dual-reporter assessment in combination with Fluc. The Gluc blood assay appears to provide a reliable indicator of viable tumor burden, and the combination of a blood assay and in vivo BLI using Gluc should be promising for quantifying and localizing the tumors.     

The effect of detergents on firefly luciferase reactions

The reaction rate of ATP-limited firefly luciferase-catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several-fold with a sharply defined optimum concentration of detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non-ionic and zwitterionic detergents increase reaction rate over a broad range of detergent concentrations. Enzyme stability during the reaction is not affected by non-ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates of some chemiluminescent reactions, have little effect on firefly luciferase activity. Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents are present.     

Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase

The continuous bioluminescent assay of ATP has been adapted to the study of Mg²⁺-dependent ATPases, including the (Na⁺,K⁺) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg²⁺-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.     

Removal of twelve C-terminal amino acids from firefly luciferase abolishes activity

cDNA coding for the luciferase in the firefly Photinus pyralis was cloned using pcDV1 primer and Honjo linker containing SP6 RNA polymerase promoter. This enabled conditions to be established to produce mRNA, capped with m7 GpppG, in vitro and then translated to form light emitting protein. Full length recombinant luciferase produced by in vitro translation, was fully active, had the same isoelectric focusing point as the native enzyme and produced a similar, yellow emission. Removal of the coding sequence for the last 12 amino acids at the C terminus, containing the peroxisome signal peptide, by polymerase chain reaction resulted in greater than or equal to 99% loss in activity of the protein formed from mRNA in vitro. This has important implications for using this luciferase as an indicator or reporter gene in eukaryotic cells, and for identifying the active centre of the enzyme.     

Nucleoside triphosphate specificity of firefly luciferase

Twelve naturally occurring nucleoside triphosphates have been examined as substrates and inhibitors of the light-producing reaction of firefly luciferase. Deoxyadenosine 5'-triphosphate was 1.7% as effective relative to ATP as a substrate, whereas all others tested were less than 0.1% as effective as ATP. At concentrations normally present in mammalian cell extracts no interference with ATP measurements results from these nucleotides.     

Atypical kinetics of immobilized firefly luciferase

The kinetic properties of collagen-bound firefly luciferase have been investigated. Under definite hydrodynamic conditions with low agitation in the reaction medium, the observed behavior is modified compared to the enzyme free in solution: reducing the stirring rate decreases the observed enzymatic activity. But diffusional resistances alone cannot account for these atypical kinetics though mass transfer may certainly play an important role during the transient state of the bioluminescent reaction. After immobilization, the time necessary to reach the steady state increased from 300 ms to 3 min and the two substrates, luciferin and ATP, behave differently with respect to the enzyme: The nature of the saturating substrate first in contact with the bound enzyme is not indifferent suggesting that immobilization can reveal behaviors or mechanisms which are not visualized with the free enzyme.     

The carboxyl-terminal tripeptide serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast.

Firefly luciferase is imported into peroxisomes in insects, mammals, plants, and yeast, which implies that the mechanism of protein translocation into peroxisomes has been conserved during eukaryotic evolution. The carboxyl-terminal tripeptide serine-lysine-leucine in luciferase acts as a peroxisomal import signal in mammalian cells. We have investigated whether this tripeptide is also involved in translocation of firefly luciferase into peroxisomes in yeast (Saccharomyces cerevisiae). We show by gene fusion experiments that the carboxyl-terminal 104 amino acids of luciferase can direct a heterologous protein to yeast peroxisomes. Luciferase mutant proteins were tested for their ability to be imported into yeast peroxisomes in vivo. We demonstrate that mutations in the carboxyl-terminal serine-lysine-leucine tripeptide abolish translocation of the protein into yeast peroxisomes. However, when a passenger protein was tagged at its carboxyl terminus with this tripeptide the fusion protein did not go to peroxisomes. These results indicate that, in yeast, the tripeptide is necessary but not sufficient for peroxisomal import.     

The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation

Here a screening method is described for transformed tissues and transgenic plants of Dendrobium (Orchidaceae) using the firefly luciferase gene ( luc ) as a combined marker/reporter gene. Protocorm-likebodies (PLB) were bombarded with tungsten particles (1.3 µm) coated with plasmids carrying a 35S-luc chimeric gene. Three weeks after bombardment 1 mM luciferin was added to the tissues and transformed cells were identified by virtue of their bioluminescence as monitored by low-light video microscopy in combination with a real-time photon imaging technique. Transformed tissues were excised, allowed to proliferate, and then subjected to a second round of screening. After three rounds of growth and screening, transformed Dendrobium tissues expressing luciferase were used to generate transgenic plants. Southern blot analysis of several transgenic lines confirmed the integration of the luciferase gene into the orchid genome. It is thought that this procedure can be used for transformation of not only orchids but other species as well.     

Efficient singlet oxygen inactivation of firefly luciferase

Firefly luciferase is inactivated by singlet oxygen at near diffusion controlled rates, 1.9 X 10(9) M-1 s-1, based on direct comparison with the oxidation of L-histidine. The inactivation kinetics are multiphasic. Inactivation is inhibitable by NaN3. Surface-separated-sensitizer (SSS) system in which singlet oxygen is produced above an air gap separating the reaction solution from the Rose Bengal sensitizer, ensuring only Type II reactions, was compared with a Sensitox II system in which the polymer bound Rose Bengal is contained in the reaction solution and both Type I and Type II reactions can occur. A slight stabilization is afforded by MgSO4.     

Inhibition of firefly luciferase by alkane analogues

We reported that anesthetics increased the partial molal volume of firefly luciferase (FFL), while long-chain fatty acids (LCFA) decreased it. The present study measured the actions of dodecanol (neutral), dodecanoic acid (negatively charged), and dodecylamine (positively charged) hydrophobic molecules on FFL. The interaction modes are measured by (1) ATP-induced bioluminescence of FFL and (2) fluorescence of 2-(p-toluidino)naphthalene-6-sulfonate (TNS). TNS fluoresces brightly in hydrophobic media. It competes with the substrate luciferin on the FFL binding. From the Scatchard plot of TNS titration, the maximum binding number of TNS was 0.83, and its binding constant was 8.27 x 10(5) M(-1). Job's plot also showed that the binding number is 0.89. From the TNS titration of FFL, the binding constant was estimated to be 8.8 x 10(5) M(-1). Dodecanoic acid quenched the TNS fluorescence entirely. Dodecanol quenched about 25% of the fluorescence, whereas dodecylamine increased it. By comparing the fluorescence of TNS and bioluminescence of FFL, the binding modes and the inhibition mechanisms of these dodecane analogues are classified in three different modes: competitive (dodecanoic acid), noncompetitive (dodecylamine), and mixed (dodecanol).     

Monoclonal antibodies assisting refolding of firefly luciferase

The reactivation efficiency in the refolding of denatured luciferase in the presence and the absence of monoclonal antibodies (mAbs) has been studied. Luciferase could be partially reactivated when the protein was denatured in high concentrations of guanidium chloride (GdmCl; >4.5 M) and the refolding was carried out in very low protein concentrations. The refolding yield was, however, significantly lower when it was performed on luciferase that had been denatured with lower concentrations of GdmCl. The efficiency of refolding decreases when the formation of aggregates increases. Three of the five luciferase mAbs tested (4G3, N2E3, S2G10) dramatically increased the yield of reactivation and simultaneously eliminated the formation of aggregates. It is proposed that these mAbs assisted the refolding of luciferase by binding to the exposed hydrophobic surface of the refolding intermediate, thus preventing it from aggregating. The epitopes interacting with these refolding-assisting mAbs are all located in the A-subdomain of the N-terminal region of luciferase. These results have also shed light on the structural features of the intermediate and its interface involved in protein aggregate formation, contributing to the understanding of the protein folding mechanism.