Luciferin-luciferase assay

Figure 3. Influence at 500 (lM salicylic acid on ATP content of excised oat roots at four pH values. ATP determined by luciferin/ luciferase assay.

Andre et al. [8] discuss the determination of adenosine-5 -triphosphate by luciferin-luciferase assay. This method was applied to the determination of adenosine-5 -triphosphate in bacterial colonies filtered from samples of polluted water after incubation for different periods. The adenosine-5 -triphosphate was extracted from the residue in the filter and the amount compared with the biochemical oxygen demand of the filtered water. The oxygen uptake rate and the rate of formation of adenosine-5 -triphosphate were then plotted against time, the two curves being similar in up to three to four days incubation, after which adenosine-5 -triphosphate production declined markedly, although oxygen uptake continued to increase. 

Tobin et al. [14] give details of two extraction procedures for the determination of adenosine-5 -triphosphate in sediment samples by luciferin-luciferase assay. 

Fig. 6.5. Time-course of ATP synthesis in a suspension of PPase, ATPase liposomes. 25 /nl liposomes (about 0.07 mg protein per ml) were suspended in a medium containing 1 ml 0.2 M glycylglycine (pH 7.8), 0.2 ml luciferin/luciferase assay, 20 fjl 100 mM sodium phosphate, 10 pi 10 mM ADP and 50 pi 10 mM sodium pyrophosphate. The final concentration of MgClj was about 10 mM. At the arrow, 25 pi liposomes were added. The resulting luminescence was measured in an LKB luminometer 1250. The light output was calibrated by addition of a known amount of ATP. (From Ref. 98.)...

Adenosine triphosphate (ATP) is produced in the course of metabolism by all living cells and, upon cell death, theoretically rapidly degrades. Thus, measurement of the compound may provide an estimate of biomass (and, hopefully, viable cell numbers). ATP released from living cells after boiling the sample in Tris buffer is measured using the luciferin-luciferase assay in which reduced luciferin is oxidized in the presence of oxygen, luciferase enzyme, ATP, and Mg ... 

The luciferin-luciferase assay commonly used to measure ATP is as follows ... 

Assays of earthworm luciferin and luciferase (Bellisario et al., 1972). The standard assay mixture contains luciferin, luciferase... 

Assay of luminescence activity. Luciferin solution (1 ml) is mixed with 1.2 ml of 0.5 M Tris buffer (pH 8.2) and 0.3 ml of luciferase solution. The luminescence reaction is initiated by the injection of 0.5 ml of 0.176 mM H2O2 to the luciferin-luciferase mixture. The light emission is characterized by a flash of light, followed by a rapid decay to a much lower steady-state level (Fig. 10.4.1). The maximum light intensity of the flash is taken as the measure of the luminescence activity. 

Enzymatic Assay. The enzymatic (luciferase) assay for adenosine triphosphate (ATP) is one of the methods applied to areas of biocidal control in oil production operation [1454]. A reliable method for the determination of ATP is the measurement of bioluminescence produced by the luciferin luciferase system. 

ATP measured by luciferin-luciferase BL assay was used to examine the effect of toxic substances on whole microbial communities in activated sludge mixed liquid samples [114], It was used to detect whether wastewater had an effect on the biodegradation capability of the resident population of microorganisms. Actually ATP BL represents an important rapid toxicity test that utilizes waste treatment natural microorganisms to determine the toxicity of wastes discharged to the sewer [132],... 

The jellyfish Aequorea contains a photoprotein, which emits light only when calcium ions are present.672 673 Since light emission can be measured with great sensitivity (modern photomultipliers can be used to count light quanta) the protein aequorin and related photoproteins674a are used as a sensitive indicator of calcium ion concentration.674 (In a similar way the firefly luciferin-luciferase system, which requires ATP for activation, is widely used in an assay for ATP.)... 

Even using these reagents some ATP may still be adsorbed onto the soil, so it is necessary to calculate the amount recovered. This is done by adding a known amount of ATP to extractant B and comparing the counts obtained with those from extractant A without added ATP. Following ultrasonics, the filtered soil extracts can be analyzed immediately or stored frozen (-18°C) for weeks or months. The ATP is assayed by the firefly luciferin-luciferase enzyme system using a bioluminometer or scintillation counter set to count out of coincidence. 

We have developed a highly sensitive simultaneous bioluminescent assay of firefly luciferase and aequorin. Firefly luciferin-luciferase reaction is specific and sensitive for the determination of ATP, and this reaction has been widely used, e.g. for hygiene monitoring. Aequorin bounds specifically to Ca and then emits blue light, thus aequorin is useful to study intercellular We thought that these photoproteins... 

In order to develop Ae PPDK-luciferin/luciferase reaction for measurement of pyrophosphate, the optimal conditions based on the presence of excess AMP were determined as PPDK luciferin-luciferase solution described in Procedure. The calibration curve of pyrophosphate was obtained via this method. Pyrophosphate from Ixio to 1x10 M was examined. The calibration curve of pyrophosphate is illustrated in Fig.2. The detection limit of pyrophosphate was 1.5 xlO mol/assay (as blank + 2SD). 

In conclusion, a novel bioluminescent pyrophosphate assay utilizing the PPDK-luciferinAuciferase reaction was established in order to measure quantitatively PCR products. Detection of pyrophosphate (1.56x10 mol/assay) was possible with the proposed method. Furthermore, this bioluminescent assay in association with allele-specific PCR was applied to the analysis of the dex gene of mutans streptococcus. The novel bioluminescent assay for PCR product based on the PPDK-luciferin/luciferase reaction appears to afford a suitable technique for diagnosis and prevention of bacterial infection and disease. 

Standard curve for NOC7. Based on the aforementioned optimum measurement conditions, NOC7 (10 13 to 10 " mol/assay) was examined. The standard curve for NOC7 is shown in Fig. 3. The detection limit of NOC7 was 10 13 mol/assay. This result was lower than the expected sensitivity of the PPDK-luciferase bioluminescent system the PPi detection limit was 10 15 mol/assay for this system.2 The reason for the reduced limit is attributable to the non-specific reaction of GTP and luciferase. The non-specific reaction of luciferin-luciferase was reflected in the presence of excess GTP consequently, the light emission intensity of the background was increased to measure NOC7 at less than 10 14 mol/assay. 

Fig. 2. Comparison between Mnz+ and Mg2+ in the novel bioluminescent assay for NO utilizing the PPDK-luciferin/luciferase system.

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