Assay of picomole amounts of ATP, ADP, and AMP using the luciferase enzyme system.

Procedural modifications of the luciferase method for ATP assay in conjunction with enzymatic conversion of AMP and ADP allow the assay of all three adenine nucleotides in quantities ranging from 4 to 20 pmoles. An unmodified Beckman scintillation detector at ambient temperature and in a coincidence mode of operation serves as a suitable instrument for quantitating light emitted by the enzyme preparation. The most significant modifications include use of Ca₃(PO₄) activated crude arsenate extracts of desiccated firefly lanterns, low arsenate concentrations during the assay, and an assay pH of 8.0. Extracts handled in this manner exhibit approximately fivefold higher activity than nonactivated extracts employed at pH 7.4 and 50 mm arsenate. Stability of activated extracts is also somewhat greater than for nonactivated preparations. ADP can be 95% enzymatically converted to ATP by treatment with phosphoenolpyruvate and pyruvate kinase under the conditions described. If myokinase is included, approximately 90% of sample AMP can be converted to ATP. Follwing the appropriate enzymatic treatment, the nucleotides are assayed as ATP and amounts calculated by comparison to curves established for known nucleotide standards. The method is appropriate for perchloric acid extracts of biological tissue and certain considerations necessary for application to experimental situations are described.     

Simple assay of 0.1–1.0 pmol of ATP,ADP, and AMP in single somatic cells using purified luciferin luciferase

The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1–1.0 pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0.1–1.0 pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described.     

A quantitative method for the measurement of cellular guanosine triphosphate pool specific activities

Studies on quantitation of RNA synthesis in eucaryotic cells have frequently used adenosine as the radioactively labeled precursor, largely because of the convenience of the firefly luciferin-luciferase assay in measuring ATP pool specific activity (1,2). This could result in some difficulties if the addition of poly(A) to the 3′ OH end of RNA represents a significant portion of total incorporation, as is the case in sea-urchin embryos (3). In addition, in some cases, the ATP pool may be large enough to prevent the use of adenosine as an effective labeling agent. Hence, a simple and sensitive method for the determination of the specific activity of the other nucleic acid precursor pools would be of value.Although the crystalline luciferase is specific for ATP, extracts of firefly lanterns most commonly used for quantitating ATP (4–9) also exhibit activity with other ribonucleoside triphosphates, adenosine tetraphosphate, ADP, and the deoxyribonucleoside triphosphates. This activity is due to the presence of contaminating enzymes such as nucleoside 5′-diphosphate kinase and adenylate kinase which catalyze the formation of ATP from these nucleotides and trace amounts of ADP, also present in the extracts (10–13). Recently, Manandhar and Van Dyke (14) have reported a procedure for quantitating picomole levels of GTP with a crude extract of firefly lanterns. In the present study, we have adapted their procedure to develop an assay for GTP pool specific activity in Xenopus laevis oocytes microinjected with [8-³H]GTP. Our assay may be extended to the analysis of any nucleoside triphosphate pool, provided that an adequate chromatography system is available for the separation of the extracted nucleotides.     

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.     

Inhibition of nuclear histone proteolysis by nucleotides]

Effects of nucleotides on the proteolysis of histones in nuclei incubated at 37 degrees C during 1, 3 and 20 h were studied. It has been shown that the H1 histone is removed first during proteolysis and then the H3 and H2B histones are digested. The H4 histone is not cleaved even after 20 h incubation. PMSF and ATP inhibit the H1 cleavage when its structure was not disturbed before ATP, CTP, ADP, NAD+, AMP and NADH inhibit the partial cleavage of the core histones H3 and H2B. ATP, CTP, AMP and NADH prevent the total digestion of H2B. ATP and, at lower extent, CTP prevent the H3 digestion. ATP, CTP, ADP and NAD+ inhibit the cleavage of the H2A histone in the experiments with 20 h incubation, when H4 is only resistant in the absence of nucleotides. The data obtained suggest an important role of ATP and other nucleotides in maintaining the structure of histones and chromatin.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Inhibition of polyoma virus middle T antigen-associated tyrosyl kinase activity by N-ethylmaleimide

The middle T antigen (MT Ag) encoded by polyoma virus has an associated protein kinase activity which transfers a phosphoryl group from ATP or GTP to a tyrosine residue on MT Ag in immunoprecipitates formed between polyoma virus-infected or transformed cell extracts and serum from animals bearing polyoma-induced tumors. Incubation of such immunoprecipitates or polyoma-transformed cell extracts prior to immunoprecipitation with the sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant inhibition of MT Ag-associated kinase activity. Inactivation of this enzyme activity by NEM was found to be dependent upon the incubation pH, time of incubation, and NEM concentration. ATP, GTP, and ADP in the presence of Mg2+ were found to decrease the rate of NEM-mediated inactivation of MT Ag-associated kinase activity, while CTP and UTP did not detectably alter the rate of enzyme inhibition by NEM. These results suggest that the MT Ag-associated kinase possesses at least one NEM-sensitive sulfhydryl group essential for phosphotransferase activity which may be present at or near the enzyme catalytic site.     

Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP.

We present methods to measure ATP, phosphocreatine, and total creatine (the sum of creatine and phosphocreatine) in alkaline cell extracts. Knowledge of these parameters, together with the known equilibrium constants for the creatine kinase and adenylate kinase-catalyzed reactions, allows one to estimate the levels of free ADP and free AMP inside cells. The enzymatic assays for the above-mentioned metabolites all lead up to the production of ATP, which is measured luminometrically with the ATP-dependent oxidation of luciferin catalyzed by firefly luciferase. To determine phosphocreatine, endogenous ATP is first destroyed, and phosphocreatine is then quantitatively reacted with exogenous ADP to form ATP. Total creatine is measured after quantitative conversion of creatine to phosphocreatine with a large excess of exogenous ATP, conversion of all ATP to ADP, and final reaction of phosphocreatine with ADP to form ATP. We used 5-microl samples in 0.5-ml microcentrifuge tubes and subsequent 5-microl additions of analytical reagents. We expect that the volumes can be changed easily. We tested the methods with glucagon- and insulin-secreting cells. Estimates of free ADP and AMP are expected to be useful in many different areas of research, such as cellular energy metabolism, purine nucleotide metabolism, adenine nucleotide gating of ion channels, and release of vasoactive or angiogenic factors.     

An apparatus for the measurement of very small membrane relaxation currents.

The reaction kinetics of crude firefly lantern extracts with and without added synthetic luciferin were examined. The addition of exogenous luciferin to the reaction mixture resulted in an apparent increase in net light emission per unit of ATP in solution. This additional reactivity (up to 1000-fold) enables the detection of subpicogram levels of ATP. The effects of enzyme preparation, dilution, and aging procedures on the increased sensitivity of the ATP assay, as well as the assay limitations of the crude firefly lantern extracts, are also discussed.     

Determination of adenosine 5'-triphosphate by the luciferase system with a stopped-flow spectrometer.

A stopped-flow spectrometer is used for ATP assay by firefly luciferase-luciferin method. It allows one to record initial rise of the light intensity and to differentiate the light produced due to the conversion of ADP to ATP by nucleoside diphosphokinase in the firefly lantern when other nucleoside triphosphates are present. Addition of luciferin (0.27 mm) to luciferase extract increases the light intensity by a factor of 50–100. This method can be used to measure ATP in the picomole range.     

Glutamine synthetase of Bacillus stearothermophilus. Regulation, site interactions, and functional information.

The action of various feedback modifiers on Bacillus stearothermophilus glutamine synthetase has been investigated by initial velocity kinetics, using the Mn2+-stimulated biosynthetic assay at 55 degrees C. The most potent inhibitors, used singly, are AMP, L-glutamine, and L-alanine. Other modifiers of significance include glycine, CTP, L-histidine, glucosamine 6-phosphate, and GDP. Marked synergism of action is observed for AMP in the presence of L-glutamine, L-histidine, ADP, or glucosamine 6-phosphate (glucosamine-6-P), and for CTP with ADP or GDP. Inhibition by saturating levels of many modifiers is either less than 100%, or is not overcome by elevated substrate levels, or both. This argues for modifier binding sites separate from substrate sites, notably in the cases of AMP, L-glutamine, glycine, L-alanine, glucosamine-6-P, and CTP. Glycine and L-alanine are Vmax inhibitors, whereas L-glutamine, glucosamine-6-P, GDP, and CTP alter the binding of L-glutamate. ADP and L-histidine apparently can compete directly with MnATP, but AMP alters Mn-ATP binding from a separate site. The action of several modifiers requires or is enhanced by bound substrates. Considerable antagonistic interaction is observed in experiments with modifier pairs, but the most potent inhibitors show synergistic or cumulative (independent) interactions. One may interpret antagonistic effects as due to (a) overlapping modifier domains, or (b) separate but antagonistically interacting sites. Either interpretation leads to a scheme for modifier-substrate and modifier-modifier site interactions in which the thermophilic enzyme must maintain and stabilize a great deal of complex functional information under extreme environmental conditions.     

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.     

Measurement of adenine nucleotides in plasma

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A ‘stop solution’ containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg⁺² and Ca⁺² that are co‐factors for many ATPases. Stop solution and blood were mixed using a two‐syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75–7.95) and Mg²⁺ (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0–15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account. Copyright © 2003 John Wiley & Sons, Ltd.     

Comparison of Methods for Extraction of Bacterial Adenine Nucleotides Determined by Firefly Assay

Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.     

Apparent synergism between amino donors for CTP synthesis in Chinese hamster fibroblasts

The synergistic effects of potential amino donors were studied in the assay of CTP synthetase in extracts of Chinese hamster fibroblasts. We found that L-glutamine was not effective as the sole amino donor, but combinations of L-glutamine with NH4HCO3, L-arginine or potassium phosphate did result in the conversion of UTP to CTP. L-arginine or potassium phosphate were also not effective when used alone, and NH4HCO3 was only slightly effective. Our studies demonstrate that the individual synergistic combinations were not additive; multiple combinations of components decreased rather than increased the formation of CTP. The synergistic combinations of L-glutamine with either NH4HCO3 or L-arginine had an absolute requirement for ATP; when ATP and PEP were absent no conversion of UTP to CTP occurred. The presence of GTP in a reaction mixture slightly increased the formation of CTP when L-glutamine and NH4HCO3 were used and substantially increased CTP formation when L-glutamine and L-arginine were used. De novo CTP synthesis was greatly reduced when nonradioactive CTP was added to an assay mixture, suggesting feedback inhibition. A TLC procedure has been developed that allows for the direct separation of UTP and CTP without requiring prior conversion to the mononucleotide or nucleoside level.     

Microsomal CTP:choline phosphate cytidylyltransferase: kinetic mechanism of fatty acid stimulation.

Fatty acids are known to cause an increase in the incorporation of radioactive choline into phosphatidylcholine. A coincident increase in membrane cytidylyltransferase activity is well documented. The purpose of the present studies was to determine the direct effects of oleic acid on the kinetic properties of membrane cytidylyltransferase. An examination of the reaction characteristics of membrane cytidylyltransferase revealed that membranes from adult rat lung contained high CTPase activity. This activity prevented the determination of reaction velocities at low CTP concentrations. The CTPase activity was blocked by the addition of ADP or ATP to the reaction. The addition of 6.0 mM ADP to the assay mixture enabled us to determine the effect of oleate on the CTP Km. Oleate (122 microM) caused a significant decrease in CTP Km for microsomal cytidylyltransferase (0.99 mM to 0.33 mM) and H-Form cytidylyltransferase (1.04 mM to 0.27 mM). Oleate did not decrease the CTP Km for L-Form cytidylyltransferase. Oleate had no effect on the choline phosphate Km in microsomal, H-Form or L-Form cytidylyltransferase. Oleate also increased the Vmax for cytidylyltransferase. The increase was dependent upon the concentration of oleate with a maximal increase of 50-60% at 100-130 microM oleate. We conclude that oleate has a direct stimulatory effect on cytidylyltransferase when it is in the active form (membrane bound or H-Form lipoprotein complex). We suggest that the kinetic effects operate synergistically with other regulatory mechanisms such as translocation or conversion of inactive to active species. The direct effect of oleate on the cytidylyltransferase may be an important regulatory mechanism when CTP concentrations are limiting.     

Effect of Nerve Growth Factor on Glucose Utilization and Nucleotide Content of Pheochromocytoma Cells (Clone PC12)

The effect of nerve growth factor (NGF) on the utilization and fate of uniformly labeled 14C glucose and on the content of several pyridine and purine nucleotides has been tested in the clonal cell line PC12. After incubation for 72 h with NGF, PC12 cells exhibit a 2.7-fold increase in glucose utilization and a 4.7-fold increase in CO2 release. During the same incubation period, all the nucleotides tested (NAD+, AMP, GMP, UDP-glucose, UDP-galactose, UDP, ADP, GDP, UTP, CTP, ATP, and GTP) underwent significant increments, varying from a minimum of 27% for ADP to a maximum of 90-120% for AMP, GMP, UDP-glucose, and UDP-galactose. These findings are discussed in connection with the trophic and differentiative effects of NGF in PC12 cells, which, in the presence of this factor, shifted from a neoplastic to a neuronal-like cell population.     

Application of a bioluminescence ATP assay in brewery wastewater treatment studies

The importance of having a rapid method for determining the viable biomass in activated-sludge wastewater treatment plants (WWTP) for process control and development is well recognized. The firefly bioluminescence ATP assay has been proposed for this purpose. Such an assay using partially purified firefly luciferase and synthetic firefly luciferin for the bioluminescence reaction, a liquid scintillation counter in the out-of-coincidence mode as luminescence detector, and a sludge ATP extraction technique involving dimethyl sulfoxide at room temperature is described. Experiments with several pure bacteria cultures were done to demonstrate the feasibility of applying this assay to activated sludge to activated sludge WWTP investigation and control. The ATP content of samples taken from various points in a 350000 gal/day brewery activated-sludge WWTP was monitored for 4.5 months. Good linear correlation between ATP and mixed-liquor suspended solids, return sludge suspended solids, and effluent suspended solids were observed. Percentage viabilities of the various sludge samples were derived from the ATP results.     

Radiochemical assays for adenylate kinase and AMP deaminase using polyethyleneimine-cellulose thin layers

Simple and specific radiochemical asays for adenylate kinase and AMP deaminase activities in crude tissue extracts are described. The radioactive substrate (AMP) is separated from the radioactive product (ADP or IMP) by chromatography on polyethyleneimine-cellulose thin layers. A rapid modification of the adenylate kinase assay is described in which samples of the reaction mixture are transferred directly to the polyethyleneimine-cellulose thin layer.