JPH11127893A - Enzymolysis of amp - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for avoiding influence of adenylate kinase (MK) which coexists in a sample on a reaction system and improving quantitativity in a method for determining a substrate and an enzyme by utilizing ADP- dependent hexokinase (ADP-HK) reaction. SOLUTION: A method for hydrolyzing AMP produced in ADP-HK reaction system by an enzyme, especially AMP deaminase, AMP nucleosidase or 5'- nucleosidase and substantially removing AMP from the reaction system, a method for substantially excluding the influence of MK which coexists in a sample on ADP-HK reaction system by the method for removing AMP, a method for hydrolyzing the AMP and a method for determining enzymatic activity and/or substrate concentration in the sample by reacting the sample with ADP-HK are disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test method used in the field of clinical tests and food tests, and more particularly to a method for measuring enzyme activity and substrate concentration in a sample. More specifically, the present invention provides an ADP
The present invention relates to a method for measuring an enzyme activity or a substrate concentration in a sample using a reaction system that causes a dependent hexokinase to act, and a reagent therefor.

[0002]

2. Description of the Related Art A thermotolerant bacterium (from Thermococcus, Pyroco
ccus-derived) from adenosine-5'-diphosphate (ADP)
Dependent hexokinase (HK) (hereinafter, ADP-dependent hexokinase is also referred to as ADP-HK) has been separated and purified. This enzyme, unlike a normal phosphorylase, does not use adenosine-5'-triphosphate (ATP) as a substrate, but specifically reacts with ADP to react with AMP and hexamonosaccharide phosphate (glucose-6). -Phosphoric acid, etc.) (reaction formula (1)).

[0003]

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Since this enzyme has high stability, it is known that it is effective for the measurement of substrates such as glucose and neutral fat and enzymes such as creatine phosphate kinase [Netherland Patent 9400836, J. Biol. Chem. (1994), 269, 17537-17
541, J. Biol. Chem. (1995), 270, 30453-30457, Ar
ch. Microbiol. (1997), 167, 217-232].

The principle of quantifying substance A using this enzyme is exemplified by a series of reaction formulas (2) to (4).

[0006]

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In this case, the finally generated NAD (P) H
Is measured by the absorbance at a wavelength of 340 nm. When a substance in a biological sample, particularly serum, is measured, AMP generated in the reaction process due to the action of adenylate kinase (MK) contained in serum. Since it reverts to ADP, accurate quantification is not possible. In order to inhibit the action of this MK, P1, P5-di (adenosine-5 ') 5-phosphate (Ap5A) has been used. However, it was difficult to completely inhibit the activity of MK with the present inhibitor alone (Clinical Chemistry Supplement, 236-255, 1987).

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide an AD
An object of the present invention is to provide a method for quantifying a substrate or an enzyme by the action of P-HK, which avoids the influence of MK coexisting in a sample as described above and improves quantification. Still another object of the present invention is to provide a method for quantitatively measuring AMP generated in a reaction system in which ADP-HK acts (hereinafter, also referred to as an ADP-HK reaction system).

[0009]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, AMP which is a direct product of the ADP-HK reaction is enzymatically hydrolyzed to coexist. The present inventors have found that the influence of MK can be avoided, and that the AMP can be quantified by quantifying a substance produced by enzymatic hydrolysis of AMP, thereby completing the present invention.

That is, the present invention is as follows. (1) A method for substantially removing AMP from an ADP-dependent hexokinase, wherein the AMP is enzymatically hydrolyzed in the reaction system. (2) the enzyme which hydrolyzes AMP is at least one selected from the group consisting of AMP deaminase, AMP nucleosidase and 5 ′ nucleotidase;
(1) The method described. (3) substantially removing AMP by the method for substantially removing AMP according to the above (1) or (2), thereby at least affecting a reaction system for reacting an ADP-dependent hexokinase with a sample; A method for substantially eliminating the influence of adenylate kinase coexisting in a sample. (4) quantifying the AMP generated in the reaction system in which ADP-dependent hexokinase acts, characterized by quantifying the substance produced by enzymatic hydrolysis of AMP generated in the reaction system in which ADP-dependent hexokinase acts. Method. (5) the enzyme, which hydrolyzes AMP, is at least one selected from the group consisting of AMP deaminase, AMP nucleosidase and 5 ′ nucleotidase;
(4) The method described. (6) The method according to the above (4) or (5), wherein the substance formed by hydrolysis is at least one selected from the group consisting of inosine-1-phosphate, adenine and adenosine. (7) AMP generated in a reaction system in which ADP-dependent hexokinase acts is enzymatically hydrolyzed, and AMP is substantially removed from the reaction system. A method for measuring enzyme activity and / or substrate concentration in a sample. (8) Quantifying a substance produced by enzymatic hydrolysis of AMP generated in a reaction system in which ADP-dependent hexokinase acts, wherein the enzyme activity in the sample is caused by allowing ADP-dependent hexokinase to act on the sample. And / or a method for measuring a substrate concentration. (9) the enzyme which hydrolyzes AMP is at least one selected from the group consisting of AMP deaminase, AMP nucleosidase and 5 ′ nucleotidase;
The method according to (7) or (8). (10) A reagent for measuring an enzyme activity and / or a substrate concentration in a sample by allowing ADP-dependent hexokinase to act on the sample, the reagent comprising an enzyme that hydrolyzes AMP as one element. (11) the enzyme which hydrolyzes AMP is at least one selected from the group consisting of AMP deaminase, AMP nucleosidase and 5 ′ nucleotidase;
The reagent according to 0).

Specific examples of the use of the ADP-HK reaction system in the present invention include substrates such as glucose, neutral fat, urea nitrogen, creatinine and 1,5 anhydroglucitol, creatine phosphate in a sample. Measurement of enzymes such as kinases and cholinesterases can be mentioned.

Samples to which the method of the present invention can be applied include:
In addition to a prepared sample in which the concentration of each component is known in advance, a biological sample in which the concentration of each component is unknown is used. As a biological sample,
Examples include body fluids such as blood, serum, and urine of a target mammal including a human. A sample collected for a clinical test is also preferably used.

The enzyme used in the present invention, which hydrolyzes AMP (hereinafter, also referred to as AMP hydrolase),
Although it is not particularly limited as long as it can hydrolyze AMP, specifically, EC No. 3.5.4.6 AMP deaminase,
EC No. 3.2.2.4 AMP nucleosidase and EC No.
3.1.3.6 5′-nucleotidase and the like, and these enzymes can be used alone or in combination of two or more. AMP deaminase includes animal tissues,
Although those derived from plants and yeasts can be used, those derived from rabbit muscle are preferably used. As the AMP nucleosidase, one derived from a microorganism such as Azotobacter vinelandii can be used, and as the 5'-nucleotidase, one derived from yeast or potato can be used.

According to the present invention, AMP produced in an ADP-HK reaction system is hydrolyzed by treating it with an AMP hydrolase such as AMP deaminase, AMP nucleosidase or 5'-nucleotidase without converting it to ADP. This is a method of substantially removing AMP from the reaction system, and a method of substantially avoiding the influence of MK coexisting in the sample on the ADP-HK reaction system by removing the AMP. Here, “substantially removes AMP” means that the AMP is degraded to such an extent that it does not act as a substrate for MK, and “the effect of MK coexisting in the sample” means This means that AMP generated by the ADP-HK reaction reverts to ADP once and becomes higher than the original value due to MK coexisting in the sample, and “the effect of MK coexisting in the sample is substantially eliminated. "Do" means "elimination of the effects of MK"
However, at least in the measurement of enzyme activity and substrate concentration,
This means that the measurement value is not affected. Further, as described above, AMP generated in the ADP-HK reaction system is enzymatically hydrolyzed, and AMP is substantially removed from the reaction system, thereby substantially eliminating the influence of MK coexisting in the sample. , AD on the sample
A method for measuring the enzyme activity and / or substrate concentration in a sample by allowing P-HK to act can be obtained.

Hereinafter, the present invention will be described in detail with reference to a measurement example of creatinine. Creatinine is a series of reaction formula (5)
It can be quantified by the measurement principle shown in (8).

[0016]

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This series of reactions is performed in a buffer solution, and includes creatinine deiminase (CRIMI), N-methylhydantoinase (NMHase), ADP-HK,
The activity of enzymes to be used, such as G6PDH (glucose-6-phosphate dehydrogenase) and G6PDH, is usually p
Select an appropriate buffer having a buffering capacity in the range of H7.5 to 8.5. Generally, a Tris buffer or the like is selected. Further, the series of reactions is carried out at an optimum temperature for the activity of the enzyme, usually at 25 to 40 ° C., and usually a final product can be obtained in 1 to 30 minutes. Sufficient amounts of various enzymes used in the series of reactions are appropriately added according to the amount of the substrate. When a biological sample such as blood is used as a sample, for example, CRIMI is 0.3 to 10 U / ml and NM
Hase is 0.1-2 U / ml, ADP-HK is 0.5-
About 10 U / ml and about 6 to 10 U / ml of G6PDH are usually added. As the divalent metal ion (M ²⁺ ) used in the series of reactions, magnesium or manganese is usually selected and used at 0.1 to 20 mM.
When MK coexists in the sample, AMP generated in the reaction process here is converted to ATP and MK coexisting in the sample.
AMP hydrolase such as AMP deaminase and AMP nucleosidase is added in order to prevent conversion into ADP by the action of the above. The amount of these hydrolases used in the present invention varies depending on the amount of AMP generated in the ADP-HK reaction system, reaction conditions, and the like.
The amount of enzyme is 0.1 to 20 U / ml, preferably 0.1 to 20 U / ml.
About 2 to 10 U / ml is added to the reaction system. When two or more kinds are used in combination, the number can be appropriately increased or decreased.

[0018] The AMP hydrolase is added to the reaction system by itself, and the required amount of the AMP hydrolase is stable in one of the reagents used in the measurement of the enzyme activity and substrate concentration in these samples. Can be included. It is preferable that a reagent containing AMP hydrolase as one element is packaged together with a reagent such as another enzyme necessary for the reaction system in a necessary amount to form a kit, for application to clinical tests and the like. The reagent included in the reagent containing AMP hydrolase as one component or packaged separately and included as a kit varies depending on the measurement target,
In addition to AMP hydrolase (AMP deaminase, AMP nucleosidase or 5'-nucleotidase, etc.)
Since the present invention utilizes an ADP-HK reaction system, at least ADP-HK, hexose as a substrate (glucose or the like; this is not limited when the measurement target is the hexose), divalent metal ion ( Magnesium, manganese, etc.). These reagents can be included in the state of being appropriately dissolved in a buffer solution as long as stability is maintained. Further, a buffer or the like may be included as necessary.

The method of the present invention for hydrolyzing AMP by adding AMP hydrolase to the ADP-HK reaction system not only avoids the interference of MK coexisting in the sample, but also uses the following method. Can also be applied. That is, AMP generated in the reaction system can be directly quantified. Further, by utilizing a method for quantifying AMP by enzymatic hydrolysis of AMP, A
It is possible to provide a method for measuring a substrate concentration or an enzyme activity in a sample by allowing DP-HK to act, particularly for highly sensitive measurement.

When AMP deaminase is used,
For example, it can be measured by the measurement principle shown in a series of reaction formulas (9) and (10). That is, by reacting inosine-1-phosphate generated as a result of the reaction formula (9) with inosine-1-phosphate dehydrogenase, the amount of NADH can be measured. The reaction is carried out under the optimum pH condition of the enzyme used, usually in the range of pH 7 to 8.5, at the optimum temperature of the enzyme activity, usually at 25 to 40 ° C. The final product can usually be obtained in 2 to 30 minutes. A sufficient amount of the enzyme used in the series of reactions is appropriately added according to the amount of the substrate. In the series of reactions, for example, when a biological sample such as blood is used as a sample, for example, AMP
Deaminase is 0.1 to 20 U / ml (preferably 0.1 to 20 U / ml).
2-10 U / ml), IMP dehydrogenase is 0.3
Usually about 10 to 10 U / ml is added.

[0021]

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At this time, glucose-6-phosphate, which is another product produced by the action of ADP-HK, has G-6 as shown in the above-mentioned reaction formula (8).
-Double the NAD by generating NADH by the action of PDH
Since it can be detected as the amount of H, high-sensitivity measurement becomes possible.

When AMP nucleosidase is used, it can be measured, for example, by the measurement principle shown in a series of reaction formulas (11) to (15). That is, adenine generated as a result of the reaction formula (11) is converted to inosine by adenosine deaminase (reaction formula (12)), and inosine is converted to hypoxanthine by a purine nucleoside phosphorylase (reaction formula (13)). By quantifying hydrogen peroxide generated by oxidizing xanthine with xanthine oxidase (reaction formula (14)), two molecules of hydrogen peroxide can be generated from one molecule of AMP, and high-sensitivity quantification is possible. When uricase is further acted on, as shown in reaction formula (15), another molecule of hydrogen peroxide is generated from uric acid, so that the sensitivity can be increased three times in total. The reaction is carried out under the optimum pH condition of the enzyme used, usually at pH 6 to 8.5.
The reaction is carried out at a temperature optimum for the activity of the enzyme, usually at 25 to 40 ° C. The final product can usually be obtained in 2 to 30 minutes. A sufficient amount of the enzyme used in the series of reactions is appropriately added according to the amount of the substrate. In the series of reactions, for example, when a biological sample such as blood is used as a sample, for example, AMP nucleosidase is 0.1 to 2%.
0 U / ml (preferably 0.2 to 10 U / ml), adenosine deaminase is 0.1 to 10 U / ml, and purine nucleoside phosphorylase is 0.1 to 10 U / m
1, xanthine oxidase is 0.1 to 10 U / ml,
Uricase is generally added at about 0.05 to 5 U / ml. The final product, hydrogen peroxide, can be colorimetrically determined by a conventional method using peroxidase.

[0024]

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When 5 'nucleotidase is used, it can be measured, for example, by the measurement principle shown in a series of reaction formulas (16) and (17). That is, adenosine generated as a result of the reaction formula (16) is oxidized with adenosine oxidase to generate hydrogen peroxide (reaction formula (17)), and the hydrogen peroxide is colorimetrically determined by a conventional method using peroxidase. The reaction is carried out under the optimum pH condition of the activity of the enzyme used.
The reaction is usually performed at a pH of 6 to 8 and at an optimum temperature for the activity of the enzyme, usually at 25 to 40 ° C. The final product can usually be obtained in 2 to 30 minutes. A sufficient amount of the enzyme used in the series of reactions is appropriately added according to the amount of the substrate.
In the series of reactions, for example, when a biological sample such as blood is used as a sample, for example, 5 'nucleotidase is 0.1 to 20 U / ml (preferably 0.2 to 10 U / m).
l), adenosine oxidase is 0.1 to 10 U / ml
A degree is usually added.

[0026]

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[0027]

EXAMPLES Reference examples for explaining the present invention in more detail,
The present invention will be described with reference to examples, but the present invention is not limited thereto. In addition, tris (hydroxymethyl) aminomethane and N-2-
Hydroxyethylpiperazine-N'-2-ethanesulfonic acid is abbreviated as Tris and HEPES, respectively. Reference Example: Measurement of Creatinine Concentration (Conventional Method) 1) Preparation of Measurement Reagent As a reference example, a reagent having the following composition was prepared, and a conventional measurement method using an inhibitor was performed. (First reagent) Tris (buffer) 100 mM (pH 8.0) Magnesium chloride 7 mM β-NADP 2 mM CRIMI 8 U / ml NMHase 0.5 U / ml ADP-HK 5 U / ml G6PDH 5 U / ml ( Second reagent) Tris (buffer) 100 mM (pH 8.0) Glucose 50 mM ATP 10 mM Ap5A X μM

2) Measurement The concentration (X μM) of Ap5A (adenylate kinase inhibitor) in the above second reagent was 0, 42.5, 85, 17
0, 340, 680, and 1360 μM.
The Ap5A concentration in the final reaction solution was 0, 10,
It was prepared to be 20, 40, 80, 160, and 320 μM. Adenylate kinase (MK) derived from human muscle was added to an aqueous creatinine solution of about 20 mg / L for 5,0.
Samples containing and not containing 00U / ml were used as measurement samples, respectively. 240 μl of first reagent in 20 μl of sample
After incubating at 37 ° C for 5 minutes,
The absorbance A1 at 0 nm is measured. The reaction is started by adding 80 μl of the second reagent. After reacting for 5 minutes, absorbance A2 at a wavelength of 340 nm is measured again. The creatinine concentration was determined by the following equation.

[0029]

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3) Results As shown in Table 1, in the sample containing no MK, the inhibitor Ap
A value approximately equal to the creatinine concentration prepared regardless of the presence or absence of 5A was measured. On the other hand, in the sample containing MK, the measured value decreases depending on the concentration of Ap5A, and
It can be seen that the effect of K is reduced. But 32
Even at 0 μM Ap5A, the effect of MK could not be completely eliminated.

[0031]

[Table 1]

Example 1: Measurement of creatinine concentration (AM
Use of P deaminase) 1) Preparation of measurement reagent A reagent having the following composition was prepared, and AMP was prepared by the method of the present invention.
The effect of deaminase was investigated. (First reagent) Tris (buffer) 100 mM (pH 8.0) Magnesium chloride 7 mM β-NADP 2 mM CRIMI 8 U / ml NMHase 0.5 U / ml ADP-HK 5 U / ml G6PDH 5 U / ml AMP Deaminase y U / ml (second reagent) Tris (buffer) 100 mM (pH 8.0) Glucose 50 mM ATP 10 mM

2) Measurement / Results The concentration of AMP deaminase in the first reagent (yU /
ml) were changed to 0, 1, 2, 4, 8, and 16 U / ml. In addition, in addition to various concentrations of AMP deaminase,
The same operation was performed when Ap5A was added to the second reagent so that the final concentration became 10 μM. The same creatinine concentration was measured by using the same sample as the reference example and operating in the same manner as the reference example. MK (5,000 U / m
Table 2 shows the results of the measurement samples containing l).

[0034]

[Table 2]

It was found that the effect of MK can be almost completely eliminated by adding 2 U / ml of AMP deaminase. In the reference example, the addition of 10 μM Ap5A could hardly remove the effect of MK, but 1 U / m
10 μM by adding 1 AMP deaminase
It can be seen that the effect of MK can be almost completely removed even with the Ap5A concentration of
It was also found that the effect of can be eliminated.

Example 2: Measurement of creatinine concentration (AM
Use of P nucleosidase) The operation was performed using AMP nucleosidase instead of the AMP deaminase of Example 1, and the effect of AMP nucleosidase was examined. Table 3 shows the results. AMP nucleosidase also showed the same effect as AMP deaminase.

[0037]

[Table 3]

Example 3 Measurement of Creatinine Concentration Based on AMP Quantification 1) Preparation of Reagent for Measurement In order to confirm the effect of the AMP quantification method by hydrolysis of AMP of the present invention, a reagent having the following composition was prepared. (First reagent) Tris (buffer) 100 mM (pH 8.0) Magnesium chloride 7 mM CRIMI 8 U / ml NMHase 0.5 U / ml ADP-HK 5 U / ml AMP nucleosidase 5 U / ml Adenosine deaminase 5 U 5 U / ml Purine nucleoside phosphorylase 5 U / ml Xanthine oxidase 5 U / ml Ascorbate oxidase 5 U / ml Catalase 500 U / ml Triiodophenol 4 mM (second reagent) Tris (buffer) 100 mM (pH 8.0) ) Glucose 50 mM ATP 25 mM 4-aminoantipyrine 1.6 mM peroxidase 20 U / ml sodium azide 50 mM

2) Measurement / Results Samples were diluted with the same human serum based on human serum containing a creatinine concentration of about 20 mg / L to which purified MK derived from human muscle was added at 5,000 U / ml.
It was prepared to be 1,000, 2,000, 3,000, 4,000, and 5,000 U / ml. Furthermore, human blood cells were washed three times with physiological saline, and then hemolyzed in purified water to prepare a hemolyzed sample mixed with the aforementioned human serum at a ratio of 1: 9 (equivalent to 5 g / L hemoglobin). This sample was prepared by mixing hemoglobin 0 in which physiological saline and serum were mixed at a ratio of 1: 9.
g / ml sample, and hemoglobin 0, 1,
Samples at 2, 3, 4, and 5 g / L concentrations were prepared. 20
After adding 240 μl of the first reagent to the μl sample and incubating at 37 ° C. for 5 minutes, the absorbance A1 at a wavelength of 546 nm was used.
Was measured. The reaction was started by adding 80 μl of the second reagent, and after reacting for 5 minutes, the absorbance A2 at a wavelength of 546 nm was measured again. The creatinine concentration was calculated in comparison with the absorbance of the standard solution. Table 4 shows the results. As a result, the measured value is constant irrespective of the MK concentration in the sample, and the measured value is also constant in a hemolyzed sample that is known to contain MK. It is clear that the effects can be eliminated. Furthermore, when the obtained absorbance was compared with the absorbance obtained in Example 2, it was about 7
As many times as high sensitivity measurement has been realized. This result shows that the apparent molecular extinction coefficient of the used chromogen is about 3.5 of that of NADH.
The reason for this is that the amount of hydrogen peroxide is twice as large as the amount of hydrogen peroxide, and it is clear that the method of the present invention can be carried out.

[0040]

[Table 4]

Example 4: Measurement of urea nitrogen concentration Urea nitrogen concentration was measured by Method A, Method B and Method C.
Here, the method A is a method of measuring without hydrolyzing AMP, the method B is hydrolyzing AMP, and the remaining glucose-6-phosphate (G6P) is obtained after hydrolysis of AMP.
The hydrolyzate obtained by hydrolyzing P after hydrolyzing P was measured. That is, the method of the present invention corresponds to Method B and Method C. The following reagents were prepared for each method. <Method A> (First reagent) HEPES (buffer) 20 mM (pH 7.0) β-NADP 2 mM Urea amide lyase 1 U / ml ADP-HK 5 U / ml G6PDH 3 U / ml (Second reagent) Tris (buffer) 50 mM (pH 8.0) Magnesium chloride 3 mM Glucose 50 mM Potassium bicarbonate 100 mM ATP 10 mM <Method B> (First reagent) HEPES (buffer) 20 mM (pH 7.0) β- NADP 2 mM urea amide lyase 1 U / ml ADP-HK 5 U / ml G6PDH 3 U / ml AMP deaminase 5 U / ml (second reagent) Tris (buffer) 50 mM (pH 8.0) Magnesium chloride 3 mM glucose 50 mM potassium bicarbonate 100 mM ATP 10 mM <Method C> (First reagent) HEPE (Buffer) 20 mM (pH 7.0) β-NADP 2 mM Urea amide lyase 1 U / ml ADP-HK 5 U / ml AMP deaminase 5 U / ml IMP dehydrogenase 3 U / ml (Second reagent) Tris (buffer) Agent) 50 mM (pH 8.0) Magnesium chloride 3 mM Glucose 50 mM Potassium bicarbonate 100 mM ATP 10 mM

2) Measurement / Results The above A, A
Urea nitrogen was quantified by each of the methods B and C. 270 μl of the first reagent is added to 3 μl of the sample, incubated at 37 ° C. for 5 minutes, and then the absorbance is measured at a wavelength of 340 nm. The reaction is started by adding 80 μl of the second reagent. Five minutes later, the absorbance was measured again, and the urea nitrogen concentration was determined from the difference in absorbance before and after the addition of the second reagent by the following formula.

[0043]

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Table 5 shows the measurement results. As a result, the measured value of the non-hemolyzed sample was not significantly different from that of the three methods, whereas the measured value of the hemolyzed sample in the method A performed without hydrolyzing AMP was measured to be abnormally higher than that of the other two methods. ing. This is because MK coexists at a high concentration due to hemolysis.
It is considered that P was generated and measured to a high value. In addition, of AMP and G6P generated by the action of ADP-HK, a method B in which AMP is hydrolyzed and the remaining G6P is measured is compared with a method C in which AMP is hydrolyzed and the hydrolyzate is measured. In this case, it is clear that the method of the present invention is excellent in reproducibility and effective since the two measurement results are almost the same.

[0045]

[Table 5]

[0046]

[Effect of the Invention] AM generated in the ADP-HK reaction system
By enzymatically hydrolyzing P and removing P from the reaction system, the effect of MK coexisting in the sample on the ADP-HK reaction system can be eliminated without using a known inhibitor, and the AMP ADP by using hydrolase
-AMP generated in the HK reaction system can be quantified.

Claims (11)

[Claims] 1. Adenosine-5'-diphosphate (ADP)
A method for substantially removing AMP from a reaction system characterized by enzymatically hydrolyzing adenosine-5'-phosphate (AMP) generated in a reaction system in which dependent hexokinase acts. 2. The method according to claim 1, wherein the enzyme that hydrolyzes AMP is at least one selected from the group consisting of AMP deaminase, AMP nucleosidase and 5 ′ nucleotidase. 3. A sample, which substantially removes AMP by the method for substantially removing AMP according to claim 1 or 2, thereby exerting a reaction on at least an ADP-dependent hexokinase and the sample. A method for substantially eliminating the effect of adenylate kinase coexisting therein. 4. A method for producing ADP-dependent hexokinase, comprising the steps of: quantifying a substance produced by enzymatic hydrolysis of AMP produced in an ADP-dependent hexokinase;
A method for quantifying MP. 5. The method according to claim 4, wherein the enzyme that hydrolyzes AMP is at least one selected from the group consisting of AMP deaminase, AMP nucleosidase and 5 ′ nucleotidase. 6. The substance produced by hydrolysis is inosine-
The method according to claim 4, wherein the method is at least one selected from the group consisting of 1-phosphate, adenine, and adenosine. 7. An enzymatic hydrolysis of AMP generated in a reaction system for causing ADP-dependent hexokinase to act,
A method for measuring an enzyme activity and / or a substrate concentration in a sample by causing ADP-dependent hexokinase to act on the sample, wherein AMP is substantially removed from the reaction system. 8. A method for quantifying a substance produced by enzymatically hydrolyzing AMP produced in a reaction system in which an ADP-dependent hexokinase acts, wherein the substance comprises AD.
A method for measuring enzyme activity and / or substrate concentration in a sample by allowing P-dependent hexokinase to act. 9. The method according to claim 7, wherein the enzyme that hydrolyzes AMP is at least one selected from the group consisting of AMP deaminase, AMP nucleosidase and 5 ′ nucleotidase. 10. A reagent for measuring an enzyme activity and / or a substrate concentration in a sample by allowing an ADP-dependent hexokinase to act on the sample, the reagent comprising an enzyme that hydrolyzes AMP as one element. 11. The method according to claim 11, wherein the enzyme that hydrolyzes AMP is AMP.
The reagent according to claim 10, which is at least one selected from the group consisting of deaminase, AMP nucleosidase and 5 'nucleotidase.