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

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

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10⁴ colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP_i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP_i-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.     

Chemiluminescent assay of co-factors

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperox-idase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 × 10^?14 to 5 × 10^?12mol/assay. The detection limit of NADH was 30fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.     

Immobilization of luciferase from a firefly lantern extract on glass strips as an alternative strategy for luminescent detection of ATP

The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution, luciferase is unstable and cannot be re-used, a problem that can be partially circumvented by immobilizing the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilizing matrices, since it allows most of the emitted photons to be detected. We report a new method for luciferase immobilization on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation. Our method is based on the co-immobilization by adsorption of luciferase (from a firefly lantern extract) and poly-L -lysine (PL) on non-porous glass strips. Luciferase immobilized in this way exhibits minimal variations in intersample activity, high sensitivity for ATP detection (linear luminescence responses down to 50 nmol/L) and good stability (full activity for at least 60 days when stored at −80°C). PL-mediated immobilization of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research. © 1998 John Wiley & Sons, Ltd.     

基于ATP生物发光法的微生物数量快速检测技术的研究进展

微生物数量的快速检测一直是工业生产与食品行业需要解决的问题,腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)生物发光法具有操作简便、检测周期短等优点,可满足一般微生物检测的需求。然而,ATP生物发光法的准确性也受到不同因素的影响,如微生物的ATP检测限值较高、微生物自身及其他因素(如非微生物ATP、提取剂种类、荧光素酶活性等)均对微生物数量的检测产生影响。本文简述了不同微生物数量检测方法的优缺点,介绍了ATP生物发光法的发展历程及原理,综述了非微生物ATP与游离ATP、微生物量、ATP提取剂、荧光素酶等因素对ATP生物发光法灵敏度与稳定性的影响,归纳总结了ATP生物发光法及检测设备在食品、医疗、污水处理等领域的应用现状,并就ATP生物发光法体系的优化及ATP在线检测的应用等方面进行了展望,以期为ATP生物发光法的高效应用提供新的思路。     

Presence of an enzyme mediating transfer of phosphate from thiamine triphosphate to ADP in germinating maize

The presence of an enzyme involved in ATP synthesis by transfer of phosphate from thiamine triphosphate to ADP in maize germ axis was indicated by the assays on partially purified (27-fold) enzyme with luciferase method, spectrophotometric assay with hexokinase and glucose-6-phosphate dehydrogenase, and paper chromatography. Optimal activity was found at pH 9.0. The enzyme was heat-labile SH-enzyme, and its activity required the presence of Mg²⁺.     

Single bacterial cell detection using a mutant luciferase

Previously, we constructed a genetically modified luciferase of Photinus pyralis that generates more than 10-fold higher luminescence intensity than the wild-type enzyme. In this study, we demonstrate that this modified luciferase enables us to detect ATP at 10⁻¹⁸ mol, which is almost equal to the quantity contained in a single bacterial cell. Consequently,we have been able to detect bacterial contamination in samples as low as one colony-forming unit (c.f.u.) for both Escherichia coli and Bacillus subtilis.     

A sensitive assay for the reverse reaction of the first histidine biosynthetic enzyme.

A method capable of specifically detecting low levels of adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17) is given. It is based on conversion of the usual product of the enzyme, N¹-(5′-phospho-d-ribosyl)adenosine triphosphate to ATP, which is detected by luciferase luminescence utilizing a liquid scintillation spectrophotometer. The assay is linear in enzyme concentration down to a lower limit of at least 55 pg enzyme/0.2 ml.     

Enzymatic Treatment to Eliminate the Extracellular ATP for Improving the Detectability of Bacterial Intracellular ATP

A novel and effective treatment of biological samples with a combination of adenosine phosphate deaminase and apyrase was developed for reducing extracellular ATP, which has been a major problem encountered in improving the sensitivity of assays for intracellular ATP by the firefly luciferin–luciferase (L-L) method. Under the enzymatic reaction conditions, ATP and the related adenosine derivatives were converted to IMP, which are not active to the L-L system. In the model system (3.2 × 10⁻⁸mATP in 1% yeast extract solution) the treatment with adenosine phosphate deaminase resulted in the reduction of ATP to 1.3 × 10⁻¹¹m, and the concomitant use of apyrase lowered the concentration to 3.3 × 10⁻¹³m. The treatment (0.05 U/ml of adenosine phosphate deaminase and apyrase) was applied to the detection of bacteria in broth by the L-L method, affording the detection of 42 colony-forming unit (CFU)/ml ofEscherichia coliand 10 CFU/ml ofStaphylococcus aureusin the broth.     

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.     

Detection of living <Emphasis Type="Italic">Salmonella</Emphasis> cells using bioluminescence

ATP-based bioluminescence using mutant firefly luciferase was combined with an immunochromatographic lateral flow test strip assay for Salmonella enteritidis detection. In this combination method, the Salmonella-antibody–gold complex captured at the test line on the test strip was lysed by heat-treatment, and the ATP released from the cells was measured using mutant luciferase. This method resulted in approximately 1,000 times higher sensitivity in the detection of Salmonella (i.e. 10³ c.f.u./ml) compared to immunochromatographic lateral flow assay.     

Do rat cardiac myocytes release ATP on contraction?

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is 10-fold below the EC₅₀ values for most purinergic receptors expressed in the heart (1.5–20 µM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP. cardiomyocytes; luciferase     

ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. I. An assay using luciferin-luciferase luminescence

The great sensitivity of the luciferin-luciferase ATP detection system allows direct observation of ATP formation derived from single-turnover flashes in a thylakoid reaction mixture. The method can measure the energization threshold—the number of flashes required for the initiation of ATP formation—as well as detect post-illumination ATP formation after the last flash of a flash sequence. We describe the characteristics of this post-illumination phosphorylation which can be observed after a series of phosphorylating flashes (PIP⁺) or when the assay for ATP formation was performed in a traditional manner where the ADP and P_i were added after the flash-energization period (PIP^–).Comparing PIP⁺ yields and kinetics of the PIP⁺ decay under various treatments can give information about membrane energization events only if it is clearly established that different PIP⁺ yields and decay rates are not due to limitations of the luciferase-catalyzed reaction. Experiments showing that the PIP⁺ ATP yield and kinetics were due to membrane-limited deenergization events (proton efflux) rather than luciferase limitations include: (1) An uncoupler, nigericin, added after the last flash reduced the PIP⁺ yield, but had no effect on the luciferase reaction. (2) The kinetics of the luminescence after adding standard ATP were much faster than the PIP⁺ kinetics. (3) Valinomycin and K⁺ stimulated the PIP⁺ yield but had no influence on the luciferase reaction. (4) Lowering the pH from 8 to 7 increased both the PIP^– (an assay independent of luciferase kinetics) and the PIP⁺ ATP yields, an expected result owing to the greater endogenous buffering power encountered by the proton gradient when the external pH is 7.In spite of the last-mentioned point, the threshold flash number for ATP formation onset was the same for pH 7 and 8 (valinomycin, K⁺ present) at slow flash frequencies. This is consistent with a membrane-localized rather than a delocalized gradient. The accompanying reports (W. A. Beard, G. Chiang and R. A. Dilley, and W. A. Beard and R. A. Dilley,J. Bioenerg. Biomembr.) show that different conditions can lead to observing either localized or delocalized proton gradient coupling in the PIP⁺ event and the ATP onset threshold flash number.     

Continuous-flow bioluminescent dtermination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25°C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.     

An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega’s luc2 for reporter and imaging applications.     

Immobilization of luciferase from the firefly Luciola mingrelica — Catalytic properties and stability of the immobilized enzyme

Firefly (Luciola mingrelica) luciferase [Photinus luciferin 4-monooxygenase (ATP-hydrolysing); Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolysing), EC 1.13.12.7] has been immobilized on albumin and polyacrylamide gel, on AH-, CH- and CNBr-Sepharose 4B as well as on Ultragel, Ultradex and cellophane film activated by cyanogen bromide. Only immobilization on cyanogen bromide-activated polysaccharide carriers resulted in highly active immobilized luciferase. Kinetic properties of immobilized luciferase hardly differed from those of the soluble enzyme. The inactivation rate constants of soluble and immobilized luciferase were measured at pH 5.5–9.0 and 25°C as well as at pH 7.8 and 20–40°C. The ΔH^≠ and ΔS^≠ values for inactivation of soluble and immobilized luciferases were obtained. A 1000-fold stabilization effect was noted for the luciferase immobilized on CNBr-Sepharose 4B at pH 7.5 and 25°C. A stabilization mechanism for the immobilized luciferase is discussed.     

Collagen strip with immobilized luciferase for ATP bioluminescent determination

Firefly luciferase from Photinus pyralis has been covalently bound to a collagen strip via an acylazide activation process. Immobilization performed in the presence of both substrates ATP and luciferin allows to increase the activity retained on the strip. The best activity exhibited by immobilized luciferase was obtained in a 0.05M Tris-acetate buffer, pH 7.75. The pH optimum and the activation energy of luciferase have been found unchanged after immobilization. In the chosen stirring conditions, no diffusional limitations of substrates appear. ATP measurements can be performed with collagen-bound luciferase in the range 1.10(-11) M-3.10(-6) M. It was possible to store the strips at 4 degrees C in a dehydrated form; then, the bound enzyme retains 20% of its initial activity after eight months. Human blood ATP was measured with this collagen-bound luciferase and the results were found in good agreement with those obtained by soluble luciferase.     

A novel direct homogeneous assay for ATP citrate lyase

ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg²⁺ as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [¹⁴C]acetyl-CoA without detecting [¹⁴C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [¹⁴C]citrate and other substrates/cofactors CoA, ATP, and Mg²⁺, 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [¹⁴C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.     

Evidence for a fatty acid reductase catalyzing the synthesis of aldehydes for the bacterial bioluminescent reaction. Resolution from luciferase and dependence on fatty acids

The enzyme responsible for the stimulation by ATP AND NADPH of light emission catalyzed by bacterial luciferase has been partially purified from extracts of the luminescent bacterium, Photobacterium phosphoreum. The stimulatory activity was found to be stabilized by high concentrations of mercaptoethanol, permitting it to be separated from luciferase into an active and stable form and enabling further characterization of its functional properties. The activity of the enzyme was shown to be dependent not only on ATP and NADPH but also on the presence of a long chain fatty acid, and was inhibited by the addition of NADH and horse liver alcohol dehydrogenase. The specificity for fatty acids, as measured by the stimulation of luciferase activity, had a very limited range, with maximal luminescence being obtained with myristic acid and lower responses being observed only with tridecanoic and pentadecanoic acid. These results provide evidence in vitro for an enzyme in bioluminescent bacteria that functions as a fatty acid reductase converting fatty acids to aldehydes which in turn can be utilized by luciferase in the light-emitting reaction.     

Transport of adenine nucleotides in the mitochondria of Saccharomyces cerevisiae: Interactions between the ADP/ATP carriers and the ATP-Mg/Pi carrier

The ADP/ATP and ATP-Mg/Pi carriers are widespread among eukaryotes and constitute two systems to transport adenine nucleotides in mitochondria. ADP/ATP carriers carry out an electrogenic exchange of ADP for ATP essential for oxidative phosphorylation, whereas ATP-Mg/Pi carriers perform an electroneutral exchange of ATP-Mg for phosphate and are able to modulate the net content of adenine nucleotides in mitochondria. The functional interplay between both carriers has been shown to modulate viability in Saccharomyces cerevisiae. The simultaneous absence of both carriers is lethal. In the light of the new evidence we suggest that, in addition to exchange of cytosolic ADP for mitochondrial ATP, the specific function of the ADP/ATP carriers required for respiration, both transporters have a second function, which is the import of cytosolic ATP in mitochondria. The participation of these carriers in the generation of mitochondrial membrane potential is discussed. Both are necessary for the function of the mitochondrial protein import and assembly systems, which are the only essential mitochondrial functions in S. cerevisiae.