Cell-surface-localized ATP detection with immobilized firefly luciferase

We demonstrate the application of an assay technique for the detection of ATP efflux from the cell surface. Until recently, the firefly luciferase assay has conventionally been used to detect ATP release because of its high sensitivity and wide detection ability. However, since this technique can be used only to infer the amount of diffused ATP in bulk solutions, it does not accurately reflect the dynamic ATP flux at the cell membrane. We therefore constructed luciferase fusion proteins that could be immobilized onto the cell surface. However, the low activities and limited application ranges of these proteins prompted us to use biotinylated luciferase given its attributes of strong and stable localization. Furthermore, luciferase can be immobilized strongly onto the biotinylated cell surface via streptavidin-biotin binding and can thus be used to determine the dynamic release of ATP near the cell surface.     

Refolding of firefly luciferase immobilized on agarose beads

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.     

Folding of the firefly luciferase polypeptide chain with immobilized C-terminus

Refolding of Photinus pyralis firefly luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of the enzyme is fast and productive in the absence of chaperones [Svetlov et al., 2006. Protein Sci. 15, 242-247]. During cotranslational folding, the C-termini of polypeptides are bound to massive particles - ribosomes. The question arises whether the immobilization of the polypeptide C-terminus on a massive particle promotes the folding. To test this experimentally, the luciferase with oligohistidine tag at its C-terminus was prepared. This allowed us to immobilize the protein C-terminal segment on chelating Sepharose beads. Here we show that both immobilized and free chains of urea-denatured enzyme refold with the same rate. At the same time, the immobilization of luciferase results in higher refolding yield due to prevention of inter-molecular aggregation. Chaperones of the Hsp70 family promote refolding of both immobilized and free luciferase polypeptides. The results presented here suggest that the high rate of cotranslational folding is not caused by the immobilization of polypeptide C-termini by itself, but is rather due to a favorable start conformation of the growing polypeptide in the peptidyl-transferase center of the ribosome and/or the strongly vectorial character of the folding from N- to C-terminus during polypeptide synthesis.     

Folding of the firefly luciferase polypeptide chain with the immobilized C terminus

Refolding of firefly Photinus pyralis luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of luciferase is fast and productive in the absence of chaperones. During cotranslational folding, the C termini of polypeptides are associated with ribosomes, massive particles. The question arises as to whether C-terminal immobilization on a massive particle promotes folding. To study this problem experimentally, luciferase was C-tagged with hexahistidine to allow its C-terminal immobilization of chelating Sepharose. Both immobilized and free chains of the urea-denatured enzyme refolded at the same rate. At the same time, immobilization led to a higher refolding yield owing to the prevention of intermolecular aggregation. Chaperones of the Hsp70 family promoted folding of both immobilized and free luciferase polypeptides. It was assumed that the high rate of cotranslational folding is not ensured by mere immobilization of the C terminus of the polypeptide, but is rather due to a favorable start conformation of the growing peptide in the peptidyltransferase center of the ribosome and/or the vectorial character of the folding, proceeding from the N to the C end during polypeptide synthesis.     

Immobilization of firefly luciferase on glass rods: Properties of the immobilized enzyme

Firefly luciferase has been covalently linked with glutaraldehyde to alkylamine glass beads which had been cemented to glass rods. The immobilized enzyme has a lower pH optima than the soluble enzyme and emits light with a major peak at 615 nm, while the soluble enzyme emits light with a peak at 562 nm. The immobilized enzyme is stable and can be used for multiple assays. The peak light intensity is linear with respect to ATP concentration in the range of 1 × 10⁻⁵ to 1 × 10⁻⁸ . The luciferase rods have been used in a coupled assay to measure the rate of ATP production catalyzed by creatine phosphokinase. This immobilized luciferase should be very useful for assaying low levels of ATP in any type of sample.     

Resveratrol inhibits firefly luciferase

The potential therapeutic value of resveratrol in age-related disease settings including cancer, diabetes, and Alzheimer's has emerged from a rapidly growing body of experimental evidence. Protection from oxidative stress appears to be a common feature of resveratrol that may be mediated through SirT1, though more specific molecular mechanisms by which resveratrol mediates its effects remain unclear. This has prompted an upsurge in cell-based mechanistic studies, often incorporating reporter assays for pathway elucidation in response to resveratrol treatment. Here, we report that resveratrol potently inhibits firefly luciferase with a K(i) value of 2microM, and caution that this confounding element may lead to compromised data interpretation.     

Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.     

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.     

Factors affecting the kinetics of light emission from crude and purified firefly luciferase.

Crude and purified firefly luciferase have been used to assay ATP from 0.2 pmol to 2 μmol. Over this range of ATP concentrations, there is a large change in the kinetics of light emission. At the lowest concentrations of ATP, light emission rises to a maximum and remains constant for a minute or longer. As the concentration of ATP is increased, the peak light intensity increases and the decay rate of light increases significantly. This is true for both the crude as well as the purified enzyme. High concentration of sodium arsenate as well as other salts inhibit the peak light emission and prevent the decay in light intensity which is due to product inhibition. It is possible to obtain almost any type of kinetics by manipulating the experimental conditions.     

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).     

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.     

Administration of immobilized firefly luciferase for quantitative estimation of ATP and enzymes synthesizing and destroying ATP]

A possibility of using immobilized Luciola mingrelica luciferase for quantitative test of ATP and ATP synthetizing and degrading enzymes activities is demonstrated. A kinetic scheme is given, and experimental conditions for using L. mingrelica luciferin-luciferase system for kinetic enzyme analysis are determined.     

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.     

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.