Development of a novel telomerase assay using the PPDK–luciferin–luciferase detection system

Telomerase participates in malignant transformation or immortalization of cells, and has attracted attention as an anticancer drug screening and diagnostic tumor marker. We developed a novel telomerase assay called the PPDK–luciferin–luciferase system bioluminescence assay (PLLBA) using pyruvate phosphate dikinase (PPDK). In this assay, pyrophosphate produced by the telomerase reaction and polymerase chain reaction (PCR) is converted to ATP by PPDK, and ATP is detected by the firefly luciferin–luciferase reaction. In this work, telomerase substrate was obtained in accordance with the telomeric repeat amplification protocol (TRAP). Telomerase‐positive (500 cells/assay), ‐inactive (heated for 10 min at 85 °C) and ‐negative (only Chaps lysis buffer) samples were used. As a result, the findings clearly showed that the signal‐to‐noise (S/N) ratio of the positive cells was 39.5. After the telomerase reaction and PCR, PLLBA was completed ~ 120 s later. A high level of reproducibility was obtained with ‐ coefficient of variation (CV) of 4.1% (positive cells). The detection limit for cells using telomerase was one cell per assay. This assay for telomerase activity was also shown to be adaptable to human cancer‐derived cell lines. Copyright © 2013 John Wiley & Sons, Ltd.     

An enzymatic cycling assay for nicotinic acid adenine dinucleotide phosphate using NAD synthetase

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer.     

Validation of a rapid, large-scale assay to quantify ATP concentration in spermatozoa

Quantification of ATP content in spermatozoa is a useful assay for evaluating sperm function; however, most detection methodology relies on assessing single samples. We have developed and validated a highly repeatable assay that permits simultaneous measurement of up to 78 samples. A key feature of this assay includes combination of a phosphatase inhibition and ATP extraction step that permits maximal detection of ATP and sample storage at -20 degrees C prior to assay. The assay was validated for spermatozoa from three different species, including turkey, rooster and boar. The sensitivity of the assay differed between avian and mammalian spermatozoa, with 2.5 x 10(6) spermatozoa being the lowest number of turkey and rooster spermatozoa that could be assayed compared to 2.5 x 10(5) boar spermatozoa. Concentrations of ATP in fresh turkey semen ranged from 2.14 to 15.6 nmol/10(9) spermatozoa; similarly, freshly collected rooster semen contained from 2.16 to 21.4 nmol ATP/10(9) spermatozoa. Evaluation of turkey semen that had been stored at 4 degrees C for 24 h revealed a decline in ATP concentrations (2.35 +/- 0.34 nmol ATP/10(9) spermatozoa). Likewise, cryopreserved rooster spermatozoa contained lower concentrations of ATP (0.05 +/- 0.01 nmol ATP/10(9) spermatozoa) than non-stored spermatozoa. Boar spermatozoa contained similar concentrations of ATP, whether fresh (74.2 +/- 8.1 pmol ATP/10(6) spermatozoa), stored for 1 day (77.0 +/- 8.1 pmol ATP/10(6) spermatozoa) or 5 days (81.96 +/- 8.1 pmol ATP/10(6) spermatozoa). For all three species, assay variation was low (inter-assay, 0.66-1.9% CV; intra-assay, 1.3% CV).     

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.     

Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification,characterization, gene cloning,and applications

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.     

Reduction of nicotinamide adenine dinucleotide by pyruvate:lipoate oxidoreductase in anaerobic, dark-grown Rhodospirillum rubrum mutant C.

Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation.     

Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphate greatly depresses the signal in vitro and that micromolar concentrations of inoragnic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP-binding to luciferase. Luciferse-injected cells exposed to extracellular luciferin concentrations above about 100 μmol/1 (corresponding to a cytoplasmic level of c. 5–10 μmol/1 because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumlation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100μmol/1 extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4 μmol/1.     

Novel bioluminescent assay of alkaline phosphatase using adenosine-3'-phosphate-5'-phosphosulfate as substrate and the luciferin-luciferase reaction and its application

This paper describes a novel bioluminescent assay of alkaline phosphatase (ALP) utilizing ATP-sulfurylase and the luciferin-luciferase reaction. The principle governing the assay is as follows. Adenosine-3'-phosphate-5'-phosphosulfate, which serves as the substrate for ALP, is hydrolyzed enzymatically to produce adenosine-5'-phosphosulfate (APS). APS is converted into ATP by ATP-sulfurylase in the presence of pyrophosphate. The ATP produced is detected by the luciferin-luciferase reaction. The measurable range was 1 zmol to 100 fmol/assay and the detection limit at blank+3 SD was 10 zmol/assay. The coefficient of variation (CV, n=5) was examined at each point of the standard curve; the mean CV percentage was 4.47% (n=6). This assay system was applied to enzyme immunoassay of human chorionic gonadotropin and allele-specific PCR enzyme-linked immunosorbent assay of verotoxin gene using ALP as the label enzyme; 10(-2) mIU/mL hCG in urine and 5 pg of Escherichia coli O157 DNA could be assayed directly and with high sensitivity by the proposed method.     

Chemiosmotic ATP synthesis in photosynthetically inactive chromoplasts from Narcissus pseudonarcissus L. linked to a redox pathway potentially also involved in carotene desaturation

Mature chromoplasts from daffodil (Narcissus pseudonarcissus) flowers, although devoid of thylakoid structures, contain immunologically detectable alpha-subunits of ATP-synthase (H(+)-transporting ATP phosphohydrolase; EC 3.6.3.14). To show the presence of the entire functional protein complex, chromoplast membrane proteins were solubilized and reconstituted in phosphatidylcholine liposomes. The membranes were energized by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential, and the initial rate of ATP synthesis was measured with a luciferin/luciferase assay. In addition, by demonstrating NADPH-dependent ATP synthesis, we show that an NAD(P)H-dependent respiratory redox pathway in chromoplasts, previously identified as an important constituent of the carotene desaturation system, proceeds concomitant with membrane energization.     

Determination of terbutaline enantiomers in biological samples using liquid chromatography with coupled columns

The purpose of this work was to develop and validate a method for the separation and determination of the enantiomers of terbutaline in plasma and intestinal juice. Terbutaline was extracted from plasma and intestinal juice by liquid-solid extraction on small C18 cartridges. The extract was then analyzed by coupled column liquid chromatography with amperometric detection. For chiral separation a beta-cyclodextrin phase was used. The within-day variation (Cv) on spiked plasma samples was in the range 0.8-6.4% at 3.8-33.8 nmol/liter for the (-)-enantiomer, and 2.6-23.0% at 1.3-11.3 nmol/liter for the (+)-enantiomer. The between-day variation on spiked plasma samples was 5.5% at 10.7 nmol/liter and 13.6% at 4.3 nmol/liter for the (-)-and (+)-enantiomers, respectively. The within-day variation for intestinal juice was in the range 0.7-1.5% at 5.6-30.0 mumol/liter for the (+)-enantiomer.     

Development and validation of an ATP method for rapid estimation of viable units in lyophilised BCG Danish 1331 vaccine

An assay for quantifying viability in BCG vaccine by determining intracellular ATP content was developed and validated. ATP content was determined by measuring bioluminescence in the presence of luciferin/luciferase. During development and validation the ATP method was compared to the conventional viable count method. A key step to obtain correlation between ATP content and CFU was found to be a period of pre-incubation in a growth medium before ATP determination. During the validation, the robustness, linearity, accuracy, precision, and range were studied. The method validation study showed that the method applied was robust and applicable to determine ATP content in lyophilised BCG for estimating viability in the BCG samples. By comparison with a conventional viable count method, a high correlation between ATP content and the viable count was found; this relationship can be applied in routine quality control to estimate viable count from the ATP content determined in a sample.     

THE ANALYSIS OF BIOLUMINESCENCES OF SHORT DURATION,RECORDED WITH PHOTOELECTRIC CELL AND STRING GALVANOMETER

1. The rapid decay of luminescence in extracts of the ostracod crustacean Cypridina hilgendorfii, has been studied by means of a photoelectric-amplifier-string galvanometer recording system. 2. For rapid flashes of luminescence, the decay is logarithmic if ratio of luciferin to luciferase is small; logarithmic plus an initial flash, if ratio of luciferin to luciferase is greater than five. The logarithmic plot of luminescence intensity against time is concave to time axis if ratio of luciferin to luciferase is very large. 3. The velocity constant of rapid flashes of luminescence is approximately proportional to enzyme concentration, is independent of luciferin concentration, and varies approximately inversely as the square root of the total luciferin (luciferin + oxyluciferin) concentration. For large total luciferin concentrations, the velocity constant is almost independent of the total luciferin. 4. The variation of velocity constant with total luciferin concentration (luciferin + oxyluciferin) and its independence of luciferin concentration is explained by assuming that light intensity is a measure of the luciferin molecules which become activated to oxidize (accompanied with luminescence) by adsorption on luciferase. The adsorption equilibrium is the same for luciferin and oxyluciferin and determines the velocity constant.     

Detection of endogenous ethanol and other compounds in the breath by gas chromatography with on-column concentration of sample

A new method is described for collecting and concentrating volatile compounds in the breath, in order to facilitate their assay by gas chromatography. Breath was collected into sealed Mylar bags containing an internal standard (isopropyl alcohol). The sample was pumped through a cooled gas chromatograph column, where the volatile compounds were concentrated by adsorption onto the resin packing (Porapak Q) at 35 degrees C. The column was then heated, and the volatilized sample was separated for assay by flame ionization detection. The assay was highly sensitive for ethanol (detecting at least 4.0 nmol) and linear up to 20 nmol (r2 = 0.98). Accuracy and precision were determined by assaying nine replicates of a sample containing 12.0 nmol ethanol; a mean value of 12.18 nmol ethanol was obtained with a coefficient of variation of 10.26%. In a group of normal volunteers, endogenous breath ethanol concentrations ranged from 2.23 to 6.51 nmol/liter. This assay provided a number of advantages over previously described methods: The use of breath collection bags enabled the collection of samples outside the laboratory. The use of an internal standard in the collection bag reduced errors that might have resulted from leakage of the specimen. An on-column concentration of the sample in the gas chromatograph eliminated the need for an additional preconcentration device, such as a cryogenic or adsorptive trapping apparatus.     

Crystal structures of an NAD kinase from Archaeoglobus fulgidus in complex with ATP, NAD, or NADP

NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 A resolution, with its substrate NAD at 3.05 A resolution, and with the product NADP in two different crystal forms at 2.45 A and 2.0 A resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2' phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.     

Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs

High field (31)P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P(2)O(7)(4-)) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 +/- 25.9 and 37.6 +/- 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 +/- 17.0 and 36.0 +/- 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with (32)P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by (31)P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.     

A sensitive and specific radiometric method for the measurement of plasma histamine in normal individuals

A radioenzymatic method for assaying histamine using histamine-N-methyltransferase from guinea pig brain was modified to increase its sensitivity, precision, and specificity. This was achieved by incorporation of a thin-layer chromatography step and use of N_α-methyl histamine as an internal standard in each sample. No prior extraction of the plasma samples is required, permitting the assay of 30 samples in 1 working day. Histamine was detected in the plasma of 17 normal volunteers, at a level of 3.4 ± 0.69 nmol/liter (range 0.82 to 4.7 nmol/liter). In 19 asthmatics, a low-peak expiratory flow rate was found to be associated with a plasma histamine concentration above this “normal” range.     

Microplate live cell assay system for early events in mechanotransduction

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.     

A column chromatographic method for determination of plasma and erythrocyte levels of inosine and hypoxanthine

A column chromatographic separation of inosine and hypoxanthine in plasma and erythrocytic samples after deproteination by ultrafiltration, adsorption of the compounds onto charcoal, elution in pyridine/ethanol solution, and filtration by celite gel is described. Sephadex G-10 was used to separate the compounds with small but different molecular weights. Inosine and hypoxanthine values were determined by enzymatic spectrophotometric assay. In arterial, coronary venous, and erythrocyte samples the mean ± SD values for inosine were calculated as 1365 ± 560 nmol/liter of plasma, 915 ± 310 nmol/liter of plasma, and 8925 ± 6720 nmol/liter of blood, respectively. The corresponding values for hypoxanthine were 2525 ± 1950 nmol/liter, 1835 ± 1315 nmol/liter, and 11090 ± 7600 nmol/liter, respectively.     

The enzymatic measurement of adenine nucleotides and P-creatine in picomole amounts

Enzymatic methods are described for the analysis of ATP, ATP + ADP, total adenylates, or P-creatine in biological samples. The methods include (i) direct fluorometric procedures for the measurement of 0.1–10 nmol using hexokinase and glucose-6-P-dehydrogenase as the indicator step; (ii) an enzymatic cycling procedure with a sensitivity of 1–50 pmol; and (iii) the measurement of light emission in the luciferin-luciferase system with a sensitivity of 0.1–80 pmol.     

Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography

A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, Δ⁴-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane—isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15–3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09–1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic—colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low.