Range Of ATP Concentrations Research Articles

Evaluation of the viability of the Bulgarian BCG vaccine by the adenosine triphosphate assay

We applied a modified assay for the measurement of the intracellular ATP content as an alternative biochemical method for estimating the viability of the BCG vaccine instead of the standard cultural microbiological assay. Eight lyophilized BCG ampoule samples (Lot 287-2 and Lot 254-2, Bulgarian BCG strain SL222 Sofia) were tested on different days. Each calibration curve was measured for the following range of ATP concentrations: 100, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.1562 pmol. The obtained results were compared to the traditional cultural colony counting method for evaluating the viability of the BCG vaccine. The NCP of Lot 287-2 (min 2.325–max 3.300) was higher in comparison to Lot 254-2 (min 1.438–max 3.330). That result corresponded to higher ATP-content registered for Lot 287-2 (min 0.409–max 1.338) than Lot 254-2 (min 0.18–max 1.104). Our results confirmed the observation made by other researchers that the modified ATP assay is a reliable, simple, and quick method for the evaluation of the viability of the BCG vaccine. Additional compiled data about all BCG strains are needed to perform studies on the correlation between the luciferin-luciferase bioluminescent ATP assay and the cultural method for viability and enumeration of the units in the lyophilized BCG vaccine.

Elucidating Events within the Black Box of Enzyme Catalysis in Energy Metabolism: Insights into the Molecular Mechanism of ATP Hydrolysis by F1-ATPase.

Oxygen exchange reactions occurring at β-catalytic sites of the FOF1-ATP synthase/F1-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. This work presents a new theory of oxygen exchange and tests it on oxygen exchange data recorded on ATP hydrolysis by mitochondrial F1-ATPase (MF1). The apparent rate constant of oxygen exchange governing the intermediate Pi-HOH exchange accompanying ATP hydrolysis is determined by kinetic analysis over a ~50,000-fold range of substrate ATP concentration (0.1-5000 μM) and a corresponding ~200-fold range of reaction velocity (3.5-650 [moles of Pi/{moles of F1-ATPase}-1 s-1]). Isotopomer distributions of [18O]Pi species containing 0, 1, 2, and 3 labeled oxygen atoms predicted by the theory have been quantified and shown to be in perfect agreement with the experimental distributions over the entire range of medium ATP concentrations without employing adjustable parameters. A novel molecular mechanism of steady-state multisite ATP hydrolysis by the F1-ATPase has been proposed. Our results show that steady-state ATP hydrolysis by F1-ATPase occurs with all three sites occupied by Mg-nucleotide. The various implications arising from models of energy coupling in ATP synthesis/hydrolysis by the ATP synthase/F1-ATPase have been discussed. Current models of ATP hydrolysis by F1-ATPase, including those postulated from single-molecule data, are shown to be effectively bisite models that contradict the data. The trisite catalysis formulated by Nath's torsional mechanism of energy transduction and ATP synthesis/hydrolysis since its first appearance 25 years ago is shown to be in better accord with the experimental record. The total biochemical information on ATP hydrolysis is integrated into a consistent model by the torsional mechanism of ATP synthesis/hydrolysis and shown to elucidate the elementary chemical and mechanical events within the black box of enzyme catalysis in energy metabolism by F1-ATPase.

A Reversible NIR‐II Ratiometric Fluorescent Probe for Real‐Time In Vivo ATP Detection

AbstractATP plays a prominent role in shaping the evolution of immune cell responses to injury, infection, and cancer. However, real‐time monitoring of ATP levels in vivo remains a challenge, due to the lack of reliable tools that work reversibly in deep tissues. Herein, based on the Förster resonance energy transfer (FRET) strategy, a reversible ratiometric second near‐infrared window (NIR‐II) molecular fluorescent probe (CX‐RATP) is designed and synthesized. CX‐RATP exhibits a significant fluorescence ratio change with the existence of 0–10 mM ATP in vitro, which is within the range of physiological and pathological ATP concentrations. Meanwhile, the recognition process can be reversed by apyrase. It can capture the increase and decrease in ATP levels in real time before and after the treatment of acute inflammation. Besides, CX‐RATP is also successfully applied to the hepatocellular carcinoma (HCC) model and achieves in situ imaging of tumor lesions. These results all provide solid proof that CX‐RATP is a powerful tool for in vivo real‐time ATP detection.

Rapid estimation of cytosolic ATP concentration from the ciliary beating frequency in the green alga Chlamydomonas reinhardtii

Determination of cellular ATP levels, a key indicator of metabolic status, is essential for the quantitative analysis of metabolism. The biciliate green alga Chlamydomonas reinhardtii is an excellent experimental organism to study ATP production pathways, including photosynthesis and respiration, particularly because it can be cultured either photoautotrophically or heterotrophically. Additionally, its cellular ATP concentration, [ATP], is reflected in the beating of its cilia. However, the methods currently used for quantifying the cellular ATP levels are time consuming or invasive. In this study, we established a rapid method for estimating cytosolic [ATP] from the ciliary beating frequency in C. reinhardtii. Using an improved method of motility reactivation in demembranated cell models, we obtained calibration curves for [ATP]–ciliary beating frequency over a physiological range of ATP concentrations. These curves allowed rapid estimation of the cytosolic [ATP] in live wild-type cells to be ∼2.0 mM in the light and ∼1.5 mM in the dark: values comparable to those obtained by other methods. Furthermore, we used this method to assess the effects of genetic mutations or inhibitors of photosynthesis or respiration quantitatively and noninvasively. This sensor-free method is a convenient tool for quickly estimating cytosolic [ATP] and studying the mechanism of ATP production in C. reinhardtii or other ciliated organisms.

A bioinspired antifouling zwitterionic interface based on reduced graphene oxide carbon nanofibers: electrochemical aptasensing of adenosine triphosphate.

An antifouling electrochemical aptasensor for ATP is described that has a zwitterionic self-assembled sensing interface on a glassy carbon electrode modified with areduced graphene oxide carbon nanofiber (GO-CNF). The GO-CNF was first modified by self-polymerization of dopamine which provided a platform for simultaneously self-assembly of the ATP aptamer and cysteine. By using hexacyanoferrate as the electrochemical probe, in the presence of ATP, the aptamer strands fold around ATP molecules, thus leading to the variation of the electrochemical signal. The aptasensor has a linear response in the 0.1 pM to 5nM ATP concentration range, and a 13 fM lower detection limit. The electrode is strongly resistant to nonspecific adsorption and biofouling. This enabled the detection of ATP even in spiked human plasma. Graphical abstract An antifouling electrochemical aptasensor employing reduced graphene oxide carbon nanofiber as conductive substrate and zwitterionic cysteine as antifouling material for adenosine triphosphate detection.

A glassy carbon electrode modified with graphene oxide, poly(3,4-ethylenedioxythiophene), an antifouling peptide and an aptamer for ultrasensitive detection of adenosine triphosphate.

An antifouling aptasensor is described for voltammetric determination ofadenosine triphosphate (ATP). A glassy carbon electrode (GCE) was modified with agraphene oxide and poly(3,4-ethylenedioxythiophene) (GO-PEDOT) composite film by electrodeposition. Next, the zwitterionic peptide (EKEKEKE) was attached. It forms an antifouling layer on themodified GCE and serves as the support for subsequent aptamer immobilization. The resulting aptasensor typically isoperated at a potential of 0.18V (vs. SCE)using hexacyanoferrate as the electrochemical probe. It has a linear response in the 0.1 pM to 1.0μM ATP concentration range, a 0.03 pM detection limit and a sensitivity of 2674.7μA·μM-1·cm-2. It has outstanding selectivity, satisfactory reproducibility and desired stability. It was used to quantify ATP in ATP-spiked 10% serum solutions. Graphical abstract Schematic presentation of the construction of the aptamer based electrode for voltammetric determination of ATP.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics.

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

Energetic Costs, Precision, and Transport Efficiency of Molecular Motors.

An efficient molecular motor would deliver cargo to the target site at a high speed and in a punctual manner while consuming a minimal amount of energy. According to a recently formulated thermodynamic principle, referred to as the thermodynamic uncertainty relation, the travel distance of a motor and its variance are, however, constrained by the free energy being consumed. Here we use the principle underlying the uncertainty relation to quantify the transport efficiency of molecular motors for varying ATP concentration ([ATP]) and applied load (f). Our analyses of experimental data find that transport efficiencies of the motors studied here are semioptimized under the cellular condition. The efficiency is significantly deteriorated for a kinesin-1 mutant that has a longer neck-linker, which underscores the importance of molecular structure. It is remarkable to recognize that, among many possible directions for optimization, biological motors have evolved to optimize the transport efficiency in particular.

A Toolbox of Chromones and Quinolones for Measuring a Wide Range of ATP Concentrations.

A series of 26 3-hydroxychromones, three bis-flavonols and four 3-hydroxyquinolones were studied to evaluate their fluorescence response to interaction with ATP in buffer. The dyes differ by the total charge, the size and number of their aromatic units, as well as the position or electron-donating ability of their substituents. All of them were suggested to form complexes with ATP of 1:1 and 1:2 stoichiometry, which can be evidenced by their bright fluorescence and their 3000-6000 cm-1 red-shifted excitation band. These fluorescent complexes allow detection of ATP concentrations over 3 orders of magnitude, whereas most other known probes cover no more than two orders. In total, the dyes allow ATP detection from 0.001 to 57 mm. In addition, most of the dye-ATP complexes can be excited in the visible and monitored in the red region of the spectrum. The response amplitude of the described dyes to ATP is as high as for the best known probes. Considering that complexation takes place at neutral pH, the studied dyes form a toolbox of fluorescent probes for intensiometric and ratiometric measurements of ATP concentration in a broad concentration range. Finally, the obtained results stimulate the idea that most of natural 3-hydroxyflavones in living cells may form complexes with ATP.

Development of a range of fluorescent reagentless biosensors for ATP, based on malonyl-coenzyme A synthetase.

The range of ATP concentrations that can be measured with a fluorescent reagentless biosensor for ATP has been increased by modulating its affinity for this analyte. The ATP biosensor is an adduct of two tetramethylrhodamines with MatB from Rhodopseudomonas palustris. Mutations were introduced into the binding site to modify ATP binding affinity, while aiming to maintain the concomitant fluorescence signal. Using this signal, the effect of mutations in different parts of the binding site was measured. This mutational analysis revealed three variants in particular, each with a single mutation in the phosphate-binding loop, which had potentially beneficial changes in ATP binding properties but preserving a fluorescence change of ~3-fold on ATP binding. Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent’s 6 μM to 123 μM and 42 μM, respectively. Kinetic measurements showed that the effect of these mutations on affinity was by an increase in dissociation rate constants. These variants widen the range of ATP concentration that can be measured readily by this biosensor to >100 μM. In contrast, a third variant, S170A, decreased the dissociation constant of ATP to 3.8 μM and has a fluorescence change of 4.2 on binding ATP. This variant has increased selectivity for ATP over ADP of >200-fold. This had advantages over the parent by increasing sensitivity as well as increasing selectivity during ATP measurements in which ADP is present.

Fluorometric aptamer based determination of adenosine triphosphate based on deoxyribonuclease I-aided target recycling and signal amplification using graphene oxide as a quencher

The authors describe an aptamer based fluorometric assay for the determination of ATP. It is based on deoxyribonuclease I-aided target recycling and signal amplification. The DNA probe consists of two regions (sequences) that represent the capture probe and the signal probe, respectively. In the absence of ATP, the probe is adsorbed by the surface of graphene oxide (GO) via π-stacking interactions. This results in quenching of the fluorescent label (carboxyfluorescein) and protects it from being cleaved by DNase. Upon adding ATP, the probe will be repelled by GO because ATP binds to the aptamer. This triggers an increase in fluorescence as measured at excitation/emission wavelengths of 480/514 nm. The detection limit is as low as 0.2 nM, and the calibration plot is linear in the 10 to 400 nM ATP concentration range. The assay is specific and sensitive, and in our perception has a large potential in terms of detecting other species including pathogenic microorganisms, small molecules, metal ions, and proteins.

Aptamer based electrochemical adenosine triphosphate assay based on a target-induced dendritic DNA nanoassembly

The authors describe an electrochemical strategy for highly sensitive determination of ATP that involves (a) aptamer-based target recognition, (b) enzyme-free dendritic DNA nanoassembly amplification with multiplex binding of the biotin-strepavidin system, and (c) enzyme-amplified differential pulse voltammetric readout. In the presence of ATP, binding of ATP to the aptamer releases trigger DNA from the double-stranded complex between ATP aptamer and trigger DNA. The single-stranded thiolated capture probe, chemisorbed on the gold electrode surface, captures the released trigger DNA via hybridization. The toehold of the trigger DNA is recombined with one end of the first substrate DNA (1) which is on its other end biotinylated and blocked, with loops, by a counterstrand. The latter is removed by a complementary single-stranded helper (1) exposing two toeholds and two identical complimentary sequences for a second biotinylated substrate DNA (2). The latter, which is double-stranded except for the toehold, binds to one of these two sites. It is then stripped from its counter strand by another single-stranded helper DNA 2, exposing a toehold to bind another substrate DNA 1. On this substrate, another cycle with dentrimeric bransching can start. Substrate 1 with its two binding sites for substrate 2 initiates the assembly of dendritic DNA on the surface of the gold electrode, which finally possesses numerous biotins at the terminal ends of both of the associated substrate DNAs. Subsequent multiplex binding of streptavidinylated alkaline phosphatase and enzyme-amplified electrochemical readout leads to a highly sensitive electrochemical ATP aptasensor. If operated in the DPV mode, the current as measured at a typical working potential of 0.25 V (vs. Ag/AgCl) increases linearly over the 10 nM to 10 μM logarithmic ATP concentration range, and the detection limit is 5.8 nM (at an S/N ratio of 3). The assay is highly specific and reproducible. It was successfully applied to the detection of ATP in spiked human serum samples.

Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

Essential Involvement of Purinergic Signaling in Calcium Handling in the Podocytes of Type 2 Diabetic Rats

Purinergic signaling (initiated via interaction of ATP with P2 receptors) is one of the major triggers of intracellular calcium influx in the epithelial cells of the glomeruli ‐ podocytes. Excessive elevation of intracellular calcium concentration in these cells results in their loss, subsequent damage of the glomeruli filtration barrier and, eventually, proteinuria. Growing evidence suggests that the activation of extracellular ATP signaling may play a crucial role in the development of diabetic nephropathy. The goal of this study was to characterize purinergic signaling in the podocytes of Type 2 Diabetic (T2D) rats. Here we used 12 week old and aged (40–60 weeks old) Wistar (control), Goto‐Kakizaki (GK) and Type 2 Diabetic Nephropathy (T2DN) rats. The GK rat is a non‐obese Wistar substrain which develops T2D early in life. T2DN rat (created by an introgression of the mitochondria and some passenger loci from the Fawn Hooded Hypertensive (FHH) rat into the background of GK rat) is a model of T2D accompanied with renal histologic abnormalities characteristic for diabetic nephropathy (DN). Using real‐time confocal microscopy we measured acute effects of ATP on intracellular calcium dynamics in the podocytes of glomeruli freshly isolated from these rat strains. We have shown that basal intracellular calcium concentration was within normal range in Wistar and GK podocytes (150.1 ± 10.8 and 174.7 ± 29.7 nM, respectively), whereas in T2DN rats it was pathologically elevated up to 232.9 ± 20.1 nM. Effect of ATP in Wistar rat podocytes (in the range of 1 to 200 μM) was dose dependent and similar to what we previously reported for Sprague Dawley rats. However, ATP‐triggered calcium influx in GK rats was substantially enhanced with dose‐response curve shifted towards stronger activation of calcium influx with lower concentrations of ATP. Importantly, the response to ATP was further increased in T2DN compared to GK rat podocytes, and was found to be dose‐independent within the same ATP concentration range. The same phenomenon was observed in aged (approximately 12 month old) GK and T2DN animals. Furthermore, experiments in calcium‐free solutions and studies using alpha, beta‐methylene‐ATP revealed a major role of extracellular calcium influx as distinct from intracellular depot depletion, as well as a likely involvement of the ionotropic P2X receptors, or calcium channels activated by P2Y‐evoked metabotropic GPCR cascades. Western blotting revealed a significantly higher expression of P2X7 receptors in the cortex of T2DN and GK strains compared to Wistar rats, and a decrease in P2Y1 expression. Thus, our data indicate that in T2DN podocytes ATP‐triggered calcium influx is enhanced compared to control animals with even low concentrations of ATP causing activation of calcium flux and augmented intracellular calcium concentration, correspondingly. This mechanism might be an important determinant of podocyte injury resulting from pathological remodeling from metabotropic P2Y to ionotropic P2X receptors occurring in DN.Support or Funding InformationSupported by ASN Ben J. Lipps fellowship, ADA 1‐15‐BS‐172, NIH DK105160 and HL122662.

Transient-State Kinetic Analysis of the RNA Polymerase I Nucleotide Incorporation Mechanism

Eukaryotes express three or more multisubunit nuclear RNA polymerases (Pols) referred to as Pols I, II, and III, each of which synthesizes a specific subset of RNAs. Consistent with the diversity of their target genes, eukaryotic cells have evolved divergent cohorts of transcription factors and enzymatic properties for each RNA polymerase system. Over the years, many trans-acting factors that orchestrate transcription by the individual Pols have been described; however, little effort has been devoted to characterizing the molecular mechanisms of Pol I activity. To begin to address this gap in our understanding of eukaryotic gene expression, here we establish transient-state kinetic approaches to characterize the nucleotide incorporation mechanism of Pol I. We collected time courses for single turnover nucleotide incorporation reactions over a range of substrate ATP concentrations that provide information on both Pol I’s nucleotide addition and nuclease activities. The data were analyzed by model-independent and model-dependent approaches, resulting in, to our knowledge, the first minimal model for the nucleotide addition pathway for Pol I. Using a grid searching approach we provide rigorous bounds on estimated values of the individual elementary rate constants within the proposed model. This work reports the most detailed analysis of Pol I mechanism to date. Furthermore, in addition to their use in transient state kinetic analyses, the computational approaches applied here are broadly applicable to global optimization problems.

Abstract 3364: Extracellular ATP-induced intracellular ATP concentration elevation mediated by macropinocytosis promotes growth, survival, and drug resistance of cancer cells

Abstract Cancer cells switch from highly efficient mitochondrial oxidative phosphorylation (OXPHOS) to low efficiency glycolysis for ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. Because of the higher rates of cell growth and proliferation, cancer cells need more ATP than normal cells of the same tissues. However, the switch is counterintuitive since cancer cells need more ATP but cancer cells prefer to use a low efficiency pathway to produce ATP. How cancer cells secure all their ATP needs is not well understood. On the other hand, extracellular ATP concentration in cancer (intratumoral ATP concentration) are found to be in the range of several hundred μM, ∼10^3-10^4 times higher than those found in normal tissues. Huge differences exist between cancer and normal cells regarding extracellular ATP. Based on these observations, we hypothesized that cancer cells take up ATP from extracellular space to supplement their energy needs. Here we report that in multiple cancer cell lines belonging to multiple cancer types, 0.5-3 mM ATP, within the intratumoral ATP concentration range, elevated intracellular ATP levels by more than 50%. Extracellular ATP also reduced stress and promoted survival of cancer cells that were under OXPHOS and glucose metabolism inhibitions. Furthermore, extracellular ATP increased the viability of cancer cells treated with sunitinib or pazopanib, anticancer drugs working as ATP analogs by competing with ATP for the ATP binding site of targeted tyrosine kinases (TKs). In contrast, extracellular ATP did not affect the activity of paclitaxel, a drug unrelated to ATP. Inhibitor studies revealed that the extracellular ATP-induced intracellular ATP increase shows no dependence on OXPHOS, glycolysis, master ATP regulator AMPK, transcription or translation. These results strongly suggest that not all the increased intracellular ATP is synthesized inside of cancer cells. Fluorescence microscopy study showed that human lung cancer A549 cells, which are KRas oncogene mutation-positive (KRas+), exhibit macropinotcytosis, a type of endocytosis that non-specifically take in extracellular nutritional molecules by “fluid drinking”. Inhibition of macropinocytosis resulted in decrease in dextran take-up and intracellular ATP levels, and alleviated the drug resistance to TKIs in dose-dependent manners. These strongly suggest that macropinocytosis is responsible, at least in part, for transporting extracellular ATP into cancer cells, which led to drug resistance. All these results demonstrate, for the first time, unrecognized roles of extracellular ATP in contributing to the intracellular ATP pool and increase drug resistance in cancer cells and suggest a novel ATP transport mechanism. These findings challenge our traditional view on ATP and shed new lights on ATP homeostasis and ATP-induced drug resistance in cancer cells. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Lingying Tong, Shiyong Wu, Xiaozhuo Chen. Extracellular ATP-induced intracellular ATP concentration elevation mediated by macropinocytosis promotes growth, survival, and drug resistance of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2014-3364

A label-free aptasensor for highly sensitive detection of ATP and thrombin based on metal-enhanced PicoGreen fluorescence

A label-free fluorescence aptasensor for highly selective and sensitive detection of ATP and thrombin was developed by using PicoGreen (PG) as signal molecule and surface-bound metal-enhanced fluorescence (MEF) substrates (silver island films, SIFs) as signal enhancers. On binding with ATP or thrombin, aptamers undergo structure switching, leading to a reduction of fluorescence intensity of PG. Chang of fluorescence intensity can be magnified by SIFs. The limit of detection for ATP and thrombin is 1.3nM and 0.073nM, respectively. The fluorescence quenching efficiency is linear in the logarithmic scale with ATP concentration range from 10nM to 100μM (R2=0.995) and thrombin concentration range from 0.1nM to 100nM (R2=0.997). The coefficients of variation of the intra-assay reproducibility and inter-assay reproducibility for ATP (10μM) assay are 3.8% and 5.2%, respectively. In addition, the aptasensor is stable and can be reliably used for ATP measurement in biological samples. Overall, the aptasensor can be a useful and cost effective tool for the specific detection of ATP, thrombin and potentially other biomolecules in biological samples.

The Reverse Gyrase from Pyrobaculum calidifontis, a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities

Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.

Prevention of Vascular Calcification by Polyphosphates and Nucleotides

In recent decades, the prevention of vascular calcification (VC) by pyrophosphate (PPi), bisphosphonates, and polyphosphates has been extensively reported. However, the possibility of direct inhibition of calcium phosphate deposition (CPD) by nucleoside-associated polyphosphates has not been addressed. We analyzed the role of ATP as an inhibitor of calcification in 2 ways: by characterizing the extracellular hydrolysis of ATP as source of PPi in the aorta, and by demonstrating the ability of ATP to prevent CPD by acting as a polyphosphate. In our study, both PPi and ATP hydrolysis in the rat aorta was kinetically characterized, thereby resulting in apparent Michaelis-Menten constants of 179 and 435 μmol/l, respectively, with the corresponding maximal velocities of 55.1 and 6,177 nmol·g(-1)·min(-1). According to these kinetic parameters, the theoretical PPi concentration in the aortic wall was 0.4-3.5 μmol/L (for an ATP concentration range of 0.1-1.0 μmol/L). In addition, we showed that nonhydrolyzable molecules are more efficient as CPD inhibitors than endogenous compounds, in accordance with the IC50 values: 1.2-2.4 μmol/L for bisphosphonates vs. 8.8 μmol/L for PPi, and 0.5-1.5 μmol/L for nonhydrolyzable ATP analogs vs. 3.2 μmol/L for ATP. Extracellular ATP can play an important role in the prevention of VC, not only as the source of PPi but also as a direct inhibitor of CPD.

A biochemical system of rapidly detecting bacteria based on ATP bioluminescence technology

A biochemical detection system based on ATP bioluminescence which can detect bacteria and microorganisms automatically and rapidly is proposed. Meanwhile, reagent formulations are optimized. In the detecting system, weak fluorescence loss is calibrated, and receiving efficiency of the photomultiplier tube is improved by making use of optical unit. The results show that rapid detection accuracy can reach 86 % compared with the traditional plate-counting method, the detecting time is controlled within twenty minutes, and detectable ATP concentration range achieves 10−15–10−8 M. Moreover, the series of curves obtained by measurements for the common variety of food (drinks, meat etc.) show that the device has a good performance and gets satisfactory testing results.