CN120888636A

CN120888636A - Reagent for detecting ADP, kit, detection method of ADP and application of reagent

Info

Publication number: CN120888636A
Application number: CN202510352914.2A
Authority: CN
Inventors: 江海清
Original assignee: Ningbo Youbo Biotechnology Co ltd
Current assignee: Ningbo Youbo Biotechnology Co ltd
Priority date: 2025-03-24
Filing date: 2025-03-24
Publication date: 2025-11-04

Abstract

This invention provides reagents, kits, methods for detecting ADP, and their applications, belonging to the field of ADP detection technology. The reagents for detecting ADP include ATP sulfurylase, pyrophosphatase, an enzyme inhibitor, an invertase that catalyzes the conversion of ADP to ATP, a substrate of the invertase, a bioluminescent enzyme, and a substrate of the bioluminescent enzyme. The enzyme inhibitor inhibits the activity of ATP sulfurylase and pyrophosphatase. This invention provides high accuracy and sensitivity in detecting ADP, with a stable fluorescence signal. This reagent can be used to detect the activity of phosphotransferases and/or ATP hydrolases.

Description

Reagent for detecting ADP, kit, detection method of ADP and application of reagent

Technical Field

The invention belongs to the technical field of ADP detection, and particularly relates to a reagent for detecting ADP, a kit, a detection method of ADP and application thereof.

Background

Phosphotransferase, also known as phosphorylase, is an enzyme that utilizes an energy molecule, such as ATP, to add phosphate groups to a corresponding substrate molecule. ATP hydrolase is an enzyme that breaks down ATP, generates ADP and phosphate groups (Pi), and releases energy.

Kinases are a phosphotransferase enzyme with important physiological functions and are mainly involved in phosphotransferase reactions in cells, which are key steps in cell signal transduction and cell metabolism. Through the catalysis of kinase, the cell can regulate the metabolic process inside the cell, and ensure the smooth progress of various biochemical reactions. Kinases, which mainly include protein kinases, lipid kinases, and glucokinases, are highly specific and catalyze only specific chemical reactions depending on the substrate class to which they are applied. They have a strong affinity for the reactants and are able to bind tightly to them, thus accelerating the reaction. At present, research on kinases has become one of the hot spots in the field of medicine research, and many medicines are designed aiming at specific kinases to regulate the activities of the kinases, so that the purpose of treating diseases is achieved. Therefore, high sensitivity, high specificity detection of kinase activity is particularly important for studying kinase function.

Methods for detecting kinase activity have been used for decades including Fluorescence Resonance Energy Transfer (FRET) assays, fluorescence Polarization (FP) assays, and radioactivity-based assays such as Scintillation Proximity Assays (SPA). FRET-based assays suffer from a number of drawbacks, including a large number of false positives, fluorescence interference of the compounds tested, narrow dynamic range and associated performance such as specificity of the antibodies used in the assay. FP assays are greatly disturbed by phosphorylated proteins and other reaction components such as lipids and detergents, thereby affecting the reproducibility of the assay. Also SPA assays are limited in their use due to factors such as the use of isotopes, the use of specific substrates, and the performance of antibodies.

In order to develop a kinase assay method with high specificity and wide application range, it is attempted to measure the ADP level in the kinase reaction or ATP hydrolase reaction by bioluminescence, thereby reflecting the activity of the relevant enzyme more accurately. For example, shultz is equal to 1993 (BioluminometricAssay ofADP AND ATP AT HIGHATP/ADP Ratios: assay ofADP afterRemoval ofATP), but the method of Shultz has 2 defects that 1) the efficiency of removing ATP by ATP sulfurylase (ATPS) in the step of removing ATP needs to be further improved, because even about 0.5% of ATP remains to interfere with the background of subsequent ADP detection, reducing the signal-to-noise ratio of the experiment, especially in a kinase reaction system with low concentration of ATP, and 2) the inactivation of ATP sulfurylase in boiling water after ATP removal not only makes the experiment operation complicated, but also makes the inactivation of boiling water inconvenient to be applied to microwell plate detection and high-throughput detection experiments.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a reagent for detecting ADP, which has high accuracy and sensitivity in detecting ADP and a stable fluorescent signal, and which can be used for detecting the enzymatic activity of phosphotransferase and/or ATP hydrolase.

In order to achieve the above object, the present invention provides the following technical solutions:

a reagent for detecting ADP, the reagent comprising ATP sulfurylase, pyrophosphatase, an enzyme inhibitor that inhibits the activity of ATP sulfurylase and pyrophosphatase, a converting enzyme that catalyzes the conversion of ADP to ATP, a substrate for the converting enzyme, a bioluminescent enzyme, and a substrate for the bioluminescent enzyme.

Preferably, the nucleotide sequence of the coding gene of ATP sulfurylase is shown as SEQ ID NO. 1.

Preferably, the invertase catalyzing ADP to ATP is any one of myokinase, creatine kinase or pyruvate kinase, and the substrate of the invertase is a phosphate donor that can be used by the invertase.

Preferably, the enzyme inhibitors include EDTA and EGTA.

It is a further object of the present invention to provide a kit for the detection of ADP comprising the above described reagents, said kit further comprising an ADP/ATP mixture standard.

It is another object of the present invention to provide a method for detecting ADP using the reagent or the kit, comprising the steps of removing ATP in a sample using ATP sulfurylase and pyrophosphatase, converting ADP in the sample into ATP using an invertase that catalyzes ADP to ATP, and detecting ATP in the sample using a bioluminescence reaction.

Preferably, the enzyme inhibitor is used to inhibit the activity of ATP sulfurylase and pyrophosphatase after removal of ATP from the sample.

It is a further object of the present invention to provide the use of said reagent or said kit or said method for detecting enzyme activity, including phosphotransferase and ATP hydrolase.

It is a further object of the present invention to provide the use of said reagent or said kit or said method for detecting and/or screening modulators of enzymatic activity, said enzymes comprising phosphotransferase and ATP hydrolase.

Preferably, the modulator includes an inhibitor of an enzyme and an activator of an enzyme.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides a reagent for detecting ADP, which can be used for qualitatively or quantitatively detecting ADP in a sample. The luminescent signal generated by detecting ADP by adopting the ADP reagent is stable and is suitable for long-time detection. The detection of ADP in a sample can be carried out by consuming substrate ATP production by phosphotransferase and/or ATP hydrolase, and the enzyme activity is positively correlated with ADP yield. Therefore, the invention can be used for detecting the enzyme activity of phosphotransferase and/or ATP hydrolase, and has the advantages of high sensitivity, good linear relation and stable fluorescent signal.

Drawings

FIG. 1 shows SDS-PAGE of purified ATP sulfurylase;

FIG. 2 is a series of standard curves for ADP/ATP mixtures at 1mM, where the left plot shows read at 30min incubation and the right plot shows stability and linearity of read over 3 h;

FIG. 3 is a 100. Mu.M series of standard curves for ADP/ATP mixtures, wherein the left plot shows read values at 30min incubation and the right plot shows stability and linearity of read values over 3 h;

FIG. 4 is a 10. Mu.M series of standard curves for ADP/ATP mixtures, wherein the left plot shows read values at 30min incubation and the right plot shows stability and linearity of read values over 3 h;

FIG. 5 is a series of standard curves for ADP/ATP mixtures at 1. Mu.M, wherein the left plot shows the read at 30min incubation and the right plot shows the stability and linearity of the read over 3 h;

FIG. 6 is a dose curve of H8-9 at ATP 10. Mu.M and EC ₅₀, where the left plot shows read at 30min incubation and the right plot shows read stability over 3H;

FIG. 7 shows the dose curve and EC ₅₀ for H8-9 at an ATP 100. Mu.M experimental condition, where the left plot shows read at 30min incubation and the right plot shows read stability over 3H;

FIG. 8 is a dose curve of PKI at ATP 10. Mu.M experimental conditions and EC ₅₀, where the left plot shows read over 30min incubation and the right plot shows read stability over 3 h;

FIG. 9 is a dose curve of PKI at ATP 100. Mu.M experimental conditions and EC ₅₀, where the left plot shows read over 30min incubation and the right plot shows read stability over 3 h;

FIG. 10 shows the dose curve and EC ₅₀ for STSP at ATP levels of 1. Mu.M and 10. Mu.M, where the left plot shows the ATP concentration at 1. Mu.M and the right plot shows the ATP concentration at 10. Mu.M.

Detailed Description

The present invention provides reagents for detecting ADP, the reagents comprising an ATP sulfurylase, a pyrophosphatase, an enzyme inhibitor that inhibits the activity of the ATP sulfurylase and pyrophosphatase, a converting enzyme that catalyzes the conversion of ADP to ATP, a substrate for the converting enzyme, a bioluminescent enzyme, and a substrate for the bioluminescent enzyme.

In the invention, the ATP sulfurylase comprises ATP sulfurylase generated by different expression systems and natural ATP sulfurylase from different species, wherein the ATP sulfurylase generated by different expression systems comprises ATP sulfurylase generated by any one of a bacterial expression system, a yeast expression system, an insect cell expression system, a mammalian cell expression system and a cell-free in-vitro expression system, and the natural ATP sulfurylase comprises any one of ATP sulfurylase in bacteria, ATP sulfurylase in fungi, ATP sulfurylase in mammals and ATP sulfurylase in plants. In the invention, the nucleotide sequence of the preferred ATP sulfurylase coding gene is shown as SEQ ID NO.1 ：ATGCCTGCTCCTCACGGTGGTATTCTACAAGACTTGATTCGTAACAAGGCGTTAAAGAAGAATGAATTGTTATCTGAAGCGCAATCTTCGGACATTTTAGTATGGAACTTGACTCCTAGACAACTATGTGATATTGAATTGATTCTAAATGGTGGGTTTTCTCCTCTGACTGGGTTTTTGAACGAAAACGATTACTCCTCTGTTGTTACAGATTCGAGATTAGCAGACGGCACATTGTGGACCATCCCTATTACATTAGATGTTGATGAAGCATTTGCTAACCAAATTAAACCAGACACAAGAATTGCCCTTTTCCAAGATGATGAAATTCCTATTGCTATACTTACTGTCCAGGATGTTTACAAGCCAAACAAAACTATCGAAGCCGAAAAAGTCTTCAGAGGTGACCCAGAACATCCAGCCATTAGCTATTTATTTAACGTTGCCGGTGATTATTACGTCGGCGGTTCTTTAGAAGCGATTCAATTACCTCAACATTATGACTATCCAGGTTTGCGTAAGACACCTGCCCAACTAAGACTTGAATTCCAATCAAGACAATGGGACCGTGTCGTAGCTTTCCAAACTCGTAATCCAATGCATAGAGCCCACAGGGAGTTGACTGTGAGAGCCGCCAGAGAAGCTAATGCTAAGGTGCTGATCCATCCAGTTGTTGGACTAACCAAACCAGGTGATATAGACCATCACACTCGTGTTCGTGTCTACCAGGAAATTATTAAGCGTTATCCTAATGGTATTGCTTTCTTATCCCTGTTGCCATTAGCAATGAGAATGAGTGGTGATAGAGAAGCCGTATGGCATGCTATTATTAGAAAGAATTATGGTGCCTCCCACTTCATTGTTGGTAGAGACCATGCGGGCCCAGGTAAGAACTCCAAGGGTGTTGATTTCTACGGTCCATACGATGCTCAAGAATTGGTCGAATCCTACAAGCATGAACTGGACATTGAAGTTGTTCCATTCAGAATGGTCACTTATTTGCCAGACGAAGACCGTTATGCTCCAATTGATCAAATTGACACCACAAAGACGAGAACCTTGAACATTTCAGGTACAGAGTTGAGACGCCGTTTAAGAGTTGGTGGTGAGATTCCTGAATGGTTCTCATATCCTGAAGTGGTTAAAATCCTAAGAGAATCCAACCCACCAAGACCAAAACAAGGTTTTTCAATTGTTTTAGGTAATTCATTAACCGTTTCTCGTGAGCAATTATCCATTGCTTTGTTGTCAACATTCTTGCAATTCGGTGGTGGCAGGTATTACAAGATCTTTGAACACAATAATAAGACAGAGTTACTATCTTTGATTCAAGATTTCATTGGTTCTGGTAGTGGACTAATTATTCCAAATCAATGGGAAGATGACAAGGACTCTGTTGTTGGCAAGCAAAACGTTTACTTATTAGATACCTCAAGCTCAGCCGATATTCAGCTAGAGTCAGCGGATGAACCTATTTCACATATTGTACAAAAAGTTGTCCTATGTCTGAAAGACAATGGCTTTTTTGTATTTTAA.

In the invention, the invertase catalyzing ADP to be ATP is any one of myokinase, creatine kinase or pyruvate kinase, the substrate of the invertase is a phosphate donor which can be used by the invertase, the invertase is preferably pyruvate kinase, and the substrate of the invertase is potassium phosphoenolpyruvate.

In the present invention, the enzyme inhibitors include EDTA (ethylenediamine tetraacetic acid) and EGTA (ethylene glycol diethylether diamine tetraacetic acid). The invention utilizes enzyme inhibitors to inactivate ATP sulfurylase and pyrophosphatase, so that the invention can be applied to microplate detection and high-throughput screening tests.

In the present invention, the bioluminescent enzyme is preferably luciferase, and the substrate of the bioluminescent enzyme is preferably D-luciferin or a D-luciferin salt.

The invention also provides a kit for detecting ADP, which comprises the reagent, and preferably, the kit further comprises an ADP/ATP mixture standard.

The invention also provides a method for detecting ADP, which uses the reagent or the kit to detect, and comprises the steps of removing ATP in a sample by using ATP sulfurylase and pyrophosphatase, converting ADP in the sample into ATP by using invertase for catalyzing the ADP to the ATP, and detecting the ATP in the sample by using bioluminescence reaction.

In the invention, when ATP exists in a sample to be detected, the accuracy of ADP detection is affected, so ATP in the sample needs to be cleared, ATP sulfurylase is utilized to convert ATP in the sample into Adenosine Monophosphate (AMP) and pyrophosphoric acid (PPi) in the presence of sodium molybdate (Na ₂MoO₄), the pyrophosphatase further catalyzes the PPi to be phosphoric acid (Pi), the pushing reaction further converts ATP into AMP, and the amount of ATP remained in the sample is reduced to 1%, 0.1%, 0.01%, 0.001%, 0.0001% or less of the initial content, or eliminated. When ATP is reduced to a minimum or eliminated, ADP is converted to ATP using a converting enzyme capable of converting ADP to ATP, and the converted ATP is then detected using a bioluminescent enzyme and a substrate for the bioluminescent enzyme.

In the present invention, preferably, the enzyme inhibitor is used to inhibit the activity of ATP sulfurylase and pyrophosphatase after ATP in the sample is removed using the ATP sulfurylase and pyrophosphatase, and the method provided by the present invention can be applied to microplate detection and high throughput screening assays.

The method provided by the invention can obviously improve the linear relation between the concentration of the compound and the signal intensity during detection, and has long half-life period of the detected signal. The detection method provided by the invention is nonradioactive, has high detection sensitivity, is convenient and fast, and can be applied to high-flux detection based on the micro-pore plate. The luminous signal generated by the invention is stable and is suitable for long-time detection. The method provided by the invention can be used for detecting the concentration of ADP or ATP in a wider range, and the invention can detect the concentration of ADP or ATP as low as 1picoM and below and detect the concentration of ADP or ATP as high as 10mM and above.

The invention also provides the application of the reagent or the kit or the method in detecting the activity of enzymes, including phosphotransferase and ATP hydrolase.

In the present invention, when the activity of phosphotransferase or ATP hydrolase is detected, the phosphotransferase or ATP hydrolase converts ATP into ADP using ATP as a substrate, and the enzyme activity is determined by detecting the amount of ADP after removing the remaining ATP in the reaction.

In the invention, when the enzyme activity is detected, ATP is mixed with phosphotransferase or ATP hydrolase to form a first mixture, the first mixture contains ADP and rest ATP, the first mixture is mixed with ATP sulfurylase and pyrophosphatase to form a second mixture, the second mixture contains AMP, pi, ATP sulfurylase, pyrophosphatase and ADP, the second mixture is mixed with enzyme inhibitor, ADP to ATP invertase substrate, bioluminescent enzyme and bioluminescent enzyme substrate to form a third mixture, and the luminous intensity of the third mixture is detected. In the present invention, the bioluminescent enzyme and the substrate for the bioluminescent enzyme may be added simultaneously with the enzyme inhibitor, ADP to ATP converting enzyme and the substrate for ADP to ATP converting enzyme, or the bioluminescent enzyme and the substrate for the bioluminescent enzyme may be added after the enzyme inhibitor, ADP to ATP converting enzyme and the substrate for ADP to ATP converting enzyme are added.

The invention also provides the use of said reagent or said kit or said method for detecting and/or screening modulators of enzyme activity, including phosphotransferase and ATP hydrolase, including inhibitors of enzymes and activators of enzymes.

In the present invention, when used for detecting and/or screening an enzyme activity modulator, ATP is mixed with a phosphotransferase or an ATP hydrolase, a phosphotransferase or an ATP hydrolase modulator to form a first mixture comprising ADP and the rest of ATP, the first mixture is mixed with an ATP sulfurylase and pyrophosphatase to form a second mixture comprising AMP, pi, ATP sulfurylase, pyrophosphatase and ADP, and the second mixture is mixed with an enzyme inhibitor, an ADP to ATP converting enzyme, a substrate for ADP to ATP converting enzyme, a bioluminescent enzyme and a substrate for bioluminescent enzyme to form a third mixture, and the luminescence intensity of the third mixture is detected. In the present invention, the bioluminescent enzyme and the substrate for the bioluminescent enzyme may be added simultaneously with the enzyme inhibitor, ADP to ATP converting enzyme and the substrate for ADP to ATP converting enzyme, or the bioluminescent enzyme and the substrate for the bioluminescent enzyme may be added after the enzyme inhibitor, ADP to ATP converting enzyme and the substrate for ADP to ATP converting enzyme are added.

Specific information on the reagents used in the specific examples of the present invention are shown in table 1.

TABLE 1 reagent information

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A reagent for detecting ADP, the reagent comprising ATP sulfurylase, pyrophosphatase, EDTA, EGTA, pyruvate kinase, phosphoenolpyruvate monopotassium salt, luciferase and D-luciferin.

ATP sulfurylase (ATPS) is an enzyme coded by an ME3 gene of Saccharomyces cerevisiae, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the gene and Ecoli expression vector pET15b are respectively subjected to double digestion by NdeI and BamH1 and then are connected to obtain a recombinant vector. Competent cells (BL 21, purchased from Sieimer's fly) were transformed with the recombinant vector, the monoclonal containing the recombinant vector was inoculated into LB agar medium, the colony was picked the next day and inoculated into 2mL of LB liquid medium, when the OD ₆₀₀ value of the medium reached 0.5, 2mL of the bacterial liquid was inoculated into 1L of LB liquid medium, when the OD ₆₀₀ value of the medium reached 2, IPTG (isopropyl-. Beta. -D-thiogalactoside) was added to the final concentration of 0.5mM, and induction was carried out for 2 hours. The culture broth was collected, centrifuged at 6000 rpm for 10 minutes in a centrifuge, and the supernatant was discarded to collect the cells. 50mL of 50mM Tris-HCl buffer (pH 7.4) was added to the cells, the cells were sonicated in a sonicator for 2 minutes, the supernatant was collected after centrifugation, and the supernatant was eluted with 200mM imidazole-containing Tris-HCl solution, followed by collection of the eluted sample. The purified protein was electrophoresed as shown in FIG. 1.

Example 2

The effect of the reagent in example 1 on removal of ATP was verified.

ATP sulfurylase is placed in 50mM Tris-HCl buffer (pH 7.4) +10mM MgCl ₂+20mMNa₂MoO₄, where the concentration of each component is the final concentration in the final solution.

ADP detection reagent 50mM Tris-HCl buffer (pH 7.4) +20mM MgSO ₄ +50mM KCl+2mM EDTA+2mM EGTA+0.1mM D-fluorescein+0.1 mM PEP-K (phosphoenolpyruvate monopotassium salt) +50 μg/mL luciferase+20U/mL pyruvate kinase, wherein the concentration of each component is the final concentration in the final solution.

The bioluminescence assay was EnVision (Perkin Elmer).

A96-well white opaque assay plate was added with 25. Mu.LATP (100. Mu.M) per well, followed by 25. Mu.L pyrophosphatase (PPA: 10U/mL) and different concentrations of ATP sulfurylase (ATPS): 2. Mu.g/mL, 1. Mu.g/mL and 0.5. Mu.g/mL.

After incubation for 40min at room temperature (22-25 ℃), 50 mu LADP detection reagent is added, and after incubation for 10min at room temperature, fluorescence values are read.

1. Mu.M ATP+0.1mM D-luciferin+50. Mu.g/mL luciferase+50 mM Tris-HCl buffer (pH 7.4) +20mM MgSO ₄, and after incubation at room temperature for 10min the fluorescence value was read as 439640.

The fluorescence was read as 47720 after incubation for 10min at room temperature with 0.1. Mu.M ATP+0.1mM D-luciferin+50. Mu.g/mL luciferase+50 mM Tris-HCl buffer (pH 7.4) +20mM MgSO ₄.

The remaining ATP after ATPS and PPA interaction can be determined by comparison with the ATP 1. Mu.M and 0.1. Mu.M fluorescence values (see Table 2). 100 μMATP was found to have about 0.006% ATP remaining under the action of ATPS (1 μg/mL) and pyrophosphatase.

TABLE 2100. Mu. MATP ATP remaining under the action of ATPS and PPA

ATPS(μg/mL)	Fluorescence value (RLU)	Residual ATP (%)
			2	2.24E+03	0.0047
1	2.88E+03	0.0060
			0.5	3.44E+03	0.0072

Example 3

ADP/ATP mixture standard curve detection experiments.

Detection was performed using the reagents of example 1.

ATP removing reagent 50mM Tris-HCl buffer (pH 7.4) +10mM MgCl ₂+20mM Na₂MoO₄ +1 μg/mLATPS +10U/mL PPA, wherein the concentration of each component is the final concentration in the reagent.

ADP detection reagent 50mM Tris-HCl buffer (pH 7.4) +20mM MgSO ₄ +50mM KCl+2mM EDTA+2mM EGTA+0.1mM D-fluorescein+0.1 mM PEP-K (phosphoenolpyruvate monopotassium salt) +50 μg/mL luciferase+20U/mL pyruvate kinase, wherein the concentration of each component is the final concentration in the reagent.

Universal kinase reaction buffer 50mM Tris buffer (pH 7.4) +20mM MgCl ₂ +0.1mg/mL BSA, where the concentration of each component is the final concentration in the buffer.

The bioluminescence assay was EnVision (Perkin Elmer).

Standard concentration of ADP/ATP mixtures the assay mimics the amount of ATP remaining and ADP produced after the kinase reaction and the ADP and ATP ratios are shown in table 3.

TABLE 3ADP and ATP ratio

%ADP	100	80	60	40	20	10	5	4	3	2	1	0
													%ATP	0	20	40	60	80	90	95	96	97	98	99	100

Standard curve concentrations of ADP/ATP mixture were configured at 1 mM:

The concentrations of ADP and ATP were diluted to 1mM with the universal kinase reaction buffer and mixed at the volumes indicated in Table 4 to give a standard concentration series of 1mM ADP/ATP mixtures.

TABLE 4ADP and ATP volume ratio

1mMADP(μL)	100	80	60	40	20	10	5	4	3	2	1	0
													1mMATP(μL)	0	20	40	60	80	90	95	96	97	98	99	100

A10-fold dilution was made with the universal kinase reaction buffer to a standard concentration series of 1mM ADP/ATP mixtures, which was 100. Mu.M ADP/ATP mixtures.

A10-fold dilution of the 100. Mu.M standard concentration series of ADP/ATP mixtures was performed with universal kinase reaction buffer to give a 10. Mu.M standard concentration series of ADP/ATP mixtures.

The standard concentration series of ADP/ATP mixture of 10. Mu.M was diluted 10-fold with the universal kinase reaction buffer to give the standard concentration series of ADP/ATP mixture of 1. Mu.M.

The test steps are as follows:

A standard concentration series of ADP/ATP mixtures of 1 mM/100. Mu.M/10. Mu.M/1. Mu.M configured as described above was added to each well of a 96-well white opaque assay plate, followed by 25. Mu.LATP removal reagent. After incubation for 40min at room temperature (22-25 ℃), 50 mu LADP detection reagent is added, and the mixture is mixed uniformly, and fluorescent values are read when incubation is carried out at room temperature for 30min, 1h, 2h, 3h, 16h and 23h respectively.

FIG. 2 shows a standard curve of a standard concentration series of 1mM ADP/ATP mixture, FIG. 3 shows a standard curve of a standard concentration series of 100. Mu.M ADP/ATP mixture, FIG. 4 shows a standard curve of a standard concentration series of 10. Mu.M ADP/ATP mixture, and FIG. 5 shows a standard curve of a standard concentration series of 1. Mu.M ADP/ATP mixture. As shown in FIGS. 2-5, the ADP concentration and the fluorescence value of the standard concentration series of the ADP/ATP mixture of 1 mM/100. Mu.M/10. Mu.M/1. Mu.M are in linear relation after being fitted by GraphPadprism 8, R ² is more than 0.99, and the fitting standard curves of the readings of the concentration series at 30min, 1h, 2h and 3h show almost coincident states, which indicates that the fluorescence intensity is kept unchanged in the 3h detection time compared with the fluorescence intensity at the time of incubation for 30 min.

In addition, the invention has the reading value of 100% of samples in the standard concentration series of 1mM ADP/ATP mixture of 1.9X10 ⁷, the reading value of 2.5X10 ⁷ in the standard concentration series of 100 mu M ADP/ATP mixture of 2.2X10 ⁶, the reading value of 1.1X10 ⁷ in the standard concentration series of 10 mu M ADP/ATP mixture, the reading value of 1.4X10 ⁶ in the standard concentration series of 100% of samples in the standard concentration series of 10 mu M ADP/ATP mixture, the reading value of 1.8X10 ⁶ in the standard concentration series of 1 mu M ADP/ATP mixture, and the reading value of 1.4X10 ⁵ in the standard concentration series of 100% of ADP/ATP mixture in the standard concentration series of 30min incubation, and the reading value of 1.8X10 ⁵ in the standard concentration series of 100% of ADP/ATP mixture in the standard concentration series of 30min incubation.

Therefore, the reading value after 16-23 h is still more than 70% of the fluorescence intensity when incubated for 30min, and R ² is more than 0.99, which shows that the invention improves the linear relation between ADP concentration and signal intensity, and the luminous signal is stable, thus being suitable for long-time detection.

Example 4

PKA (protein kinase A) specific inhibitors H8-9 and PKI were tested for PKA EC ₅₀.

Detection was performed using the reagents of example 1.

ATP removing reagent, 50mM Tris-HCl buffer (pH 7.4) +10mM MgCl ₂+20mM Na₂MoO₄ +1 μg/mLATPS +10U/mLPPA, wherein the concentration of each component is the final concentration in the reagent.

ADP detection reagent 50mM Tris-HCl buffer (pH 7.4) +20mM MgSO ₄ +50mM KCl+2mM EDTA+2mM EGTA+0.1mM D-fluorescein+0.1 mM PEP-K+50 μg/mL luciferase+20U/mL pyruvate kinase, wherein the concentration of each component is the final concentration in the reagent.

The bioluminescence assay was EnVision (Perkin Elmer).

EC ₅₀ for PKA by H8-9.

H8-9 was serially diluted 3-fold with universal kinase reaction buffer at a maximum concentration of 30. Mu.M and a minimum concentration of 1nM.

ATP concentration was 10. Mu.M:

25. Mu.L of universal kinase reaction buffer containing 80U PKA and different concentrations of H8-9 was added to each well of a 96-well white opaque assay plate, 10. Mu.MATP was added thereto, and the reaction time was 10min at room temperature (22 ℃ C. To 25 ℃ C.). After the reaction is finished, adding a 25 mu LATP removing reagent, incubating for 40min at room temperature (22-25 ℃), adding a 50 mu LADP detection reagent, uniformly mixing, and incubating for 30min, 1h, 2h, 3h and 21h at room temperature, and respectively reading fluorescence values. ADP concentration and fluorescence values were calculated by EC ₅₀ after GraphPadprism 8 fitting, and the H8-9 dose curve is shown in FIG. 6. And the read value dose curves of the concentration series at 30min, 1h, 2h and 3h show almost coincident states, which shows that the fluorescence intensity stability is strong.

The fluorescent signal of the invention is stable, the reading value after incubation for 21H is 7.5X10 ⁵ when the H8-9 concentration is 3nM, and is still more than 50% of the reading value (1.4X10 ⁶) after incubation for 30min, and EC ₅₀ is kept unchanged.

ATP concentration was 100. Mu.M:

25. Mu.L of universal kinase reaction buffer containing 80U PKA and different concentrations of H8-9 was added to each well of a 96-well white opaque assay plate, 100. Mu.MATP was added thereto, and the reaction time was 30min at room temperature (22 ℃ C. To 25 ℃ C.). After the reaction is finished, adding a 25 mu LATP removing reagent, incubating for 40min at room temperature (22-25 ℃), adding a 50 mu LADP detection reagent, uniformly mixing, and incubating for 30min, 1h, 2h, 3h and 21h at room temperature, and respectively reading fluorescence values. ADP concentration and fluorescence values were calculated by EC ₅₀ after GraphPadprism 8 fitting, and the H8-9 dose curve is shown in FIG. 7. And the read value dose curves of the concentration series at 30min, 1h, 2h and 3h show almost coincident states, which shows that the fluorescence intensity stability is strong.

The fluorescent signal of the invention is stable, the reading value after incubation for 21H is 1.8X10 ⁶ when the H8-9 concentration is 10nM, and is still more than 50% of the reading value (3X 10 ⁶) after incubation for 30min, and EC ₅₀ is kept unchanged.

EC ₅₀ of H8-9 on PKA was tested using ADP-Glo kit from Promega, and the results are shown in Table 5, where the invention is consistent with the ADP-Glo kit measurements.

TABLE 5 EC ₅₀ comparison of different methods for H8-9 detection

Pki EC ₅₀ to PKA.

PKI was serially diluted 3-fold with universal kinase reaction buffer at a maximum concentration of 1. Mu.M and a minimum concentration of 0.03nM.

ATP concentration was 10. Mu.M:

25. Mu.L of universal kinase reaction buffer containing 80UPKA and PKI at different concentrations was added to each well of a 96-well white opaque assay plate, 10. Mu.MATP was added thereto, and the reaction time was 10min at room temperature (22 ℃ C. To 25 ℃ C.). After the reaction is finished, adding a 25 mu LATP removing reagent, incubating for 40min at room temperature (22-25 ℃), adding a 50 mu LADP detection reagent, uniformly mixing, and incubating for 30min, 1h, 2h, 3h and 21h at room temperature, and respectively reading fluorescence values. ADP concentration and fluorescence values were calculated by EC ₅₀ after fitting GraphPadPrism 8 and PKI dose curves are shown in fig. 8. And the read value dose curves of the concentration series at 30min, 1h, 2h and 3h show almost coincident states, which shows that the fluorescence intensity stability is strong.

The fluorescent signal of the invention is stable, the PKI concentration is 1nM, the reading value after 21h incubation is 7.4X10 ⁵, which is still more than 50% of the reading value (1.4X10 ⁶) after 30min incubation, and EC ₅₀ is kept unchanged.

ATP concentration was 100. Mu.M:

25. Mu.L of universal kinase reaction buffer containing 80UPKA and PKI at different concentrations was added to each well of a 96-well white opaque assay plate, 100. Mu.MATP was added, and the reaction time was 30min at room temperature (22 ℃ -25 ℃). After the reaction is finished, adding a 25 mu LATP removing reagent, incubating for 40min at room temperature (22-25 ℃), adding a 50 mu LADP detection reagent, uniformly mixing, and incubating for 30min, 1h, 2h, 3h and 21h at room temperature, and respectively reading fluorescence values. ADP concentration and fluorescence values were calculated by EC ₅₀ after fitting GraphPadPrism 8 and PKI dose curves are shown in fig. 9. And the read value dose curves of the concentration series at 30min, 1h, 2h and 3h show almost coincident states, which shows that the fluorescence intensity stability is strong.

The fluorescent signal of the invention is stable, the PKI concentration is 0.03nM, the reading value after 21h incubation is 1.6X10 ⁶, the reading value is still more than 50% of the reading value (2.7X10 ⁶) after 30min incubation, and EC ₅₀ is kept unchanged.

The PKI to PKA EC ₅₀ was detected using ADP-Glo reagent from Promega corporation and the results are shown in Table 6, where the invention is consistent with the ADP-Glo reagent assay.

TABLE 6 EC ₅₀ comparison of PKI detection by different methods

Phosphotransferase (or ATP hydrolase) is capable of consuming the substrate ATP to produce the product ADP, and the activity of phosphotransferase (or ATP hydrolase) and the yield of ADP are positively correlated. The activity of the phosphotransferase or ATP hydrolase can be determined by measuring the yield of ADP after the phosphotransferase (or ATP hydrolase) reaction. If a modulator of the phosphotransferase (or ATP hydrolase) is present in the reaction when ATP is consumed by the phosphotransferase (or ATP hydrolase), the effect of the modulator on the phosphotransferase (or ATP hydrolase) can be determined by measuring the yield of ADP after the phosphotransferase (or ATP hydrolase) reaction.

The compounds H8-9 and PKI are specific inhibitors of Protein Kinase A (PKA), wherein H8-9 is a competitive inhibitor of ATP, and when the ATP concentration is increased, the inhibition of H8-9 becomes weaker, i.e. the EC ₅₀ value is significantly increased, and PKI is a non-competitive inhibitor of ATP, the EC ₅₀ and the ATP concentration of which are independent.

The consumption of substrate ATP by PKA in PKA kinase reactions produces the product ADP, the activity of which is positively correlated with the yield of ADP. PKA activity and inhibition of the kinase by H8-9 and PKI can be determined by measuring the yield of ADP after PKA kinase reaction. The invention removes the residual ATP after PKA kinase reaction through ATPS and PPA, converts the product ADP from pyruvate kinase into ATP, and simultaneously detects the quantity of the converted ATP to determine the yield of ADP.

The measurement of EC ₅₀ for H8-9 and PKI also shows that the present invention can distinguish between competitive and non-competitive inhibitors of ATP by comparing EC ₅₀ at different concentrations of ATP

Example 5

EC ₅₀ of the broad-spectrum kinase inhibitor Staurosporine (STSP) to PKA was determined.

Detection was performed using the reagents of example 1.

ADP detection reagent 50mM Tris-HCl buffer (pH 7.4) +20mM MgSO ₄ +50mM KCl+2mM EDTA+2mM EGTA+0.1mM D-fluorescein+0.1 mM PEP-K+50 μg/mL luciferase+20U/mL pyruvate kinase, wherein each component has the final concentration in the reagent.

The bioluminescence assay was EnVision (Perkin Elmer).

STSP was serially diluted 3-fold with universal kinase reaction buffer at a maximum concentration of 1. Mu.M and a minimum concentration of 0.03nM.

ATP concentration was 1. Mu.M:

25. Mu.L of universal kinase reaction buffer containing 80U PKA and STSP at different concentrations was added to each well of a 96-well white opaque assay plate, 1. Mu.MATP was added thereto, and the reaction time was 30min at room temperature (22 ℃ C. To 25 ℃ C.). After the reaction is finished, adding a 25 mu LATP removing reagent, incubating for 40min at room temperature (22-25 ℃), adding a 50 mu LADP detection reagent, uniformly mixing, and incubating for 30min at room temperature and 19h, and respectively reading fluorescence values. ADP concentration and fluorescence values were fit by GRAPHPAD PRISM and EC ₅₀ was calculated and STSP dose curves are shown in the left panel of figure 10. STSP gave an EC ₅₀ of PKA of 2.62nM at an ATP concentration of 1 μm, which was consistent with EC ₅₀ =3.51 nM as determined with ADP-Glo reagent from Promega corporation.

The fluorescent signal of the invention is stable, the reading value after incubation for 19h is 1.3X10 ⁵ when the concentration of STSP is 0.03nM, the reading value is still more than 50% of the reading value (2.2X10 ⁵) after incubation for 30min, and EC ₅₀ is kept unchanged.

ATP concentration was 10. Mu.M:

25. Mu.L of universal kinase reaction buffer containing 80U PKA and STSP at different concentrations was added to each well of a 96-well white opaque assay plate, 100. Mu.MATP was added thereto, and the reaction time was 30min at room temperature (22 ℃ C. To 25 ℃ C.). After the reaction is finished, adding a 25 mu LATP removing reagent, incubating for 40min at room temperature (22-25 ℃), adding a 50 mu LADP detection reagent, uniformly mixing, and incubating for 30min at room temperature and 19h, and respectively reading fluorescence values. ADP concentration and fluorescence values were fit by GRAPHPAD PRISM and EC ₅₀ was calculated and STSP dose curves are shown on the right hand graph in fig. 10. STSP gave an EC ₅₀ of PKA of 2.67nM at an ATP concentration of 10 μm, which is consistent with EC ₅₀ =3.63 nM as determined with ADP-Glo reagent from Promega corporation.

The fluorescent signal of the invention is stable, the reading value after incubation for 19h is 6.7X10 ⁵ when the concentration of STSP is 0.03nM, the reading value is still more than 50% of the reading value (1.2X10 ⁶) after incubation for 30min, and EC ₅₀ is kept unchanged.

In conclusion, the invention can be effectively used for detecting the activity of phosphotransferase or ATP hydrolase, and has the advantages of high sensitivity, good linear relation and stable fluorescent signal.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A reagent for detecting ADP, characterized in that the reagent comprises ATP sulfurylase, pyrophosphatase, an enzyme inhibitor, a converting enzyme catalyzing ADP to ATP, a substrate of the converting enzyme, a bioluminescent enzyme and a substrate of the bioluminescent enzyme, the enzyme inhibitor inhibiting the activity of ATP sulfurylase and pyrophosphatase.

2. The reagent according to claim 1, wherein the nucleotide sequence of the coding gene of ATP sulfurylase is shown in SEQ ID NO. 1.

3. The reagent according to claim 1, wherein the converting enzyme catalyzing ADP to ATP is any one of myokinase, creatine kinase, and pyruvate kinase, and the substrate of the converting enzyme is a phosphate donor usable by the converting enzyme.

4. The reagent of claim 1, wherein the enzyme inhibitor comprises EDTA and EGTA.

5. A kit for detecting ADP, comprising the reagent of any one of claims 1 to 4, and further comprising an ADP/ATP mixture standard.

6. A method for detecting ADP using the reagent according to any one of claims 1 to 4 or the kit according to claim 5, comprising the steps of:

Removing ATP from the sample using ATP sulfurylase and pyrophosphatase;

Converting ADP in the sample to ATP using an invertase that catalyzes the conversion of ADP to ATP;

ATP in the sample is detected using a bioluminescence reaction.

7. The method of claim 6, wherein the enzyme inhibitor is used to inhibit the activity of ATP sulfurylase and pyrophosphatase after removal of ATP from the sample.

8. Use of the reagent of any one of claims 1 to 4 or the kit of claim 5 or the method of claim 6 or 7 for detecting an enzymatic activity, wherein the enzymes comprise phosphotransferase and ATP hydrolase.

9. Use of a reagent according to any one of claims 1 to 4 or a kit according to claim 5 or a method according to claim 6 or 7 for detecting and/or screening modulators of enzymatic activity, characterized in that the enzymes comprise phosphotransferase and ATP hydrolase.

10. The use according to claim 9, wherein the modulator comprises an inhibitor of an enzyme and an activator of an enzyme.