Use of Firefly Luciferase for ATP Measurement: Other Nucleotides Enhance Turnover

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (>8μM) or a fairly constant production of light with lower concentrations of ATP (< 1 μM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.     

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.     

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5′-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP:d-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5′-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di-, and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl₂ to promote PRPP synthetase subunit aggregation. Increasing the MgCl₂ to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.     

Continous monitoring of ATP-converting reactions by purified firefly luciferase.

The time course of the bioluminescence obtained with a partially purified firefly luciferase preparation has been studied. At ATP levels less than 10^?6m the light emission could be maintained essentially constant for several minutes, if the luciferase was not subjected to product inhibition or other inactivating processes. This could be achieved by performing the reaction at appropriate pH and concentration of luciferin and luciferase. Under these conditions continuous measurement of light emission may be used for nondestructive monitoring of ATP-converting reactions, since the emission will be proportional to the ATP concentration in each instant. The continuous monitoring of ATP concentration by firefly luciferase was used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. It was found to be extremely sensitive and convenlent for routine applications.     

Two kinetically distinguishable ATP sites in firefly luciferase

Results are presented which indicate that firefly luciferase has two catalytically active sites. One site, Km of 1.1 X 10(-4) M ATP, is responsible for the initial flash and is apparently product inhibited for further light production. The second site, Km of 2 X 10(-5) M ATP, catalyzes the continuous low production of light. ATP or AMP is a potent inhibitor of the initial flash when LH2-AMP is used to initiate the light reaction but appears to have no affect on the second site low level light emission. Both sites must be occupied by ATP for the formation of one L-AMP. Thus, ATP appears to function both as a catalytically active substrate and a regulator for light emission.     

On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc.     

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.     

Site-directed mutagenesis of a residue located in the regulatory site of Escherichia coli aspartate transcarbamoylase. Involvement of lysine 94 in effector binding and the allosteric mechanism

Lysine 94 in the regulatory chain of aspartate transcarbamoylase has been changed to a glutamine residue by site-directed mutagenesis. The resulting enzyme is almost insensitive to the activator ATP and shows a substantially reduced response to the feedback inhibitor CTP. Competition experiments indicate that ATP is still able to bind at low concentrations to the regulatory site of the mutant enzyme, even though no stimulation could be detected. When the nucleosides adenosine or cytidine were used, the saturation curves of the mutant and the wild-type enzyme became indistinguishable. Together these results indicate that lysine 94 is strongly involved in the binding of ATP and CTP by interacting specifically with the triphosphate moiety of these nucleotide effectors. Furthermore, unlike the wild-type enzyme, the inhibitory and stimulatory effects in the mutant enzyme are insensitive to changes in aspartate concentrations, implying that the lysine 94 side chain is also involved in the allosteric mechanism of the enzyme.     

Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.     

Pyrophosphate and tripolyphosphate affect firefly luciferase luminescence because they act as substrates and not as allosteric effectors

The activating and stabilizing effects of inorganic pyrophosphate, tripolyphosphate and nucleoside triphosphates on firefly luciferase bioluminescence were studied. The results obtained show that those effects are a consequence of the luciferase-catalyzed splitting of dehydroluciferyl-adenylate, a powerful inhibitor formed as a side product in the course of the bioluminescence reaction. Inorganic pyrophosphate, tripolyphosphate, CTP and UTP antagonize the inhibitory effect of dehydroluciferyl-adenylate because they react with it giving rise to products that are, at least, less powerful inhibitors. Moreover, we demonstrate that the antagonizing effects depended on the rate of the splitting reactions being higher in the cases of inorganic pyrophosphate and tripolyphosphate and lower in the cases of CTP and UTP. In the case of inorganic pyrophosphate, the correlation between the rate of dehydroluciferyl-adenylate pyrophosphorolysis and the activating effect on bioluminescence only occurs for low concentrations because inorganic pyrophosphate is, simultaneously, an inhibitor of the bioluminescence reaction. Our results demonstrate that previous reports concerning the activating effects of several nucleotides (including some that do not react with dehydroluciferyl-adenylate) on bioluminescence were caused by the presence of inorganic pyrophosphate contamination in the preparations used.     

The value of Dowex-50 in fractionation of nucleotides from acid soluble pool. A caution

Problems were encountered in separating acid soluble nucleotides into four main groups by chromatography on Dowex-50(H⁺) columns. These difficulties derived from the low affinity of ATP, ADP, GDP, GTP, CDP, and CTP for Dowex-50 (H⁺) ion exchanger under mild acid conditions. Thus, under the conditions described, these compounds travel together with uridine compounds.     

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.     

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.     

The effect of ATP analogues on ATPase and phosphatase activities of Na+, K+-ATPase for duck salt glands

ATP analogues are studied for their effect on phosphatase and ATPase activities of Na+, K+-ATPase with the aim to obtain data concerning properties and structure of sites of high and low affinity to ATP. The activating effect of nucleotides on K+-dependent phosphatase reflecting their ability to be bound with the centres of high affinity decreases in a series: ATP, N1-oxy-ATP, CTP, JTP. In the domain of high ATP concentrations, where low affinity site is saturated, ADP is a competitive inhibition of ATPase reaction with Ki of 300 microM. The analysis of N1-oxy-ATP inhibiting effect has shown that its affinity to this site is six times less than that of ADP. The absence of the inhibiting effect of CDP, JDP, GDP and UDP in concentrations up to 10 mM testifies to the fact that sites of both low and high affinity to ATP are characterized by high specificity with respect to the adenine part of the substrate molecule.     

Caged ATP - an internal calibration method for ATP bioluminescence assays

ATP bioluminescence, based on the firefly luciferase system, is used for the rapid determination of hygienic practices in the food industry. This study has demonstrated the use of caged ATP as an internal ATP standard and quantified the effects of industrial cleansing solutions, alcoholic beverages and pH on firefly luciferase activity. The light signal was quenched 6-47% by five cleansing solutions at standard working concentrations. Ethanol at 1% (v/v) inhibited bioluminescence by 15% (w/v) whereas concentrations above 4% enhanced the light output. The light signal was quenched by 20-25% at pH values below pH 4 and above pH 10.     

Phosphate compounds in rat erythrocytes and reticulocytes.

The concentrations of glycolytic intermediates, including 2,3-diphosphoglycerate, were similar in rat reticulocytes and erythrocytes. There were striking differences, however, in the content and kind of water-soluble nucleotides. Reticulocytes contained much higher concentrations of ATP, GTP, UTP and CTP and had nucleotides not detected in the mature cell including UDP-acetylhexosamine, guanosine diphosphomannose and an unidentified cytidine compound. A large fraction of the total GTP found in the reticulocyte was in the form of a 1:1 complex of ferric iron with GTP.     

The chaperone function of ClpB from Thermus thermophilus depends on allosteric interactions of its two ATP-binding sites

ClpB belongs to the Hsp100 family and assists de-aggregation of protein aggregates by DnaK chaperone systems. It contains two Walker consensus sequences (or P-Loops) that indicate potential nucleotide binding domains (NBD). Both domains appear to be essential for chaperoning function, since mutation of the conserved lysine residue of the GX(4)GKT consensus sequences to glutamine (K204Q and K601Q) abolishes its properties to accelerate renaturation of aggregated firefly luciferase.The underlying biochemical reason for this malfunction appears not to be a dramatically reduced ATPase activity of either P-loop per se but rather changed properties of co-operativity of ATPase activity connected to oligomerization properties to form productive oligomers. This view is corroborated by data that show that structural stability (as judged by CD spectroscopy) or ATPase activity at single turnover conditions (at low ATP concentrations) are not significantly affected by these mutations. In addition nucleotide binding properties of wild-type protein and mutants (as judged by binding studies with fluorescent nucleotide analogues and competitive displacement titrations) do not differ dramatically. However, the general pattern of formation of stable, defined oligomers formed as a function of salt concentration and nucleotides and more importantly, cooperativity of ATPase activity at high ATP concentrations is dramatically changed with the two P-loop mutants described.     

Comparison of properties of commercially available crystalline native and recombinant firefly luciferases

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differeces are probably attributable to differences in conformation.     

Enzymatic incorporation of ATP and CTP analogues into the 3' end of tRNA

Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP):tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6, and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP):tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.     

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.