WO2016047580A1 - キナーゼを用いた新規な測定方法及び組成物 - Google Patents
キナーゼを用いた新規な測定方法及び組成物 Download PDFInfo
- Publication number
- WO2016047580A1 WO2016047580A1 PCT/JP2015/076639 JP2015076639W WO2016047580A1 WO 2016047580 A1 WO2016047580 A1 WO 2016047580A1 JP 2015076639 W JP2015076639 W JP 2015076639W WO 2016047580 A1 WO2016047580 A1 WO 2016047580A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- kinase
- nucleotide coenzyme
- phosphate
- change
- coenzyme
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01001—Hexokinase (2.7.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01011—6-Phosphofructokinase (2.7.1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/0103—Glycerol kinase (2.7.1.30)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/0104—Pyruvate kinase (2.7.1.40)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01147—ADP-specific glucokinase (2.7.1.147)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
- C12Y207/02003—Phosphoglycerate kinase (2.7.2.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
- C12Y207/03002—Creatine kinase (2.7.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
Definitions
- the present invention relates to a novel high-sensitivity measurement method by an enzyme cycling method using a kinase, and a composition thereof.
- Enzyme cycling method is a technique that amplifies and measures the concentration of a very small amount of substance using the action of an enzyme.
- a method for measuring a coenzyme a highly sensitive method for measuring a coenzyme, such as nicotinamide adenine dinucleotide (NAD) cycling using two types of dehydrogenases, is known (for example, see Non-Patent Document 1). .)
- a cycling method for measuring adenosine triphosphate (ATP) using two kinds of kinases has also been reported (see, for example, Patent Document 1).
- examples of the enzyme cycling method for measuring a substrate instead of a coenzyme include a method using two types of transferase and a method using one enzyme.
- a method using transamylase and polyamine oxidase can be mentioned (for example, see Patent Document 2).
- This method uses putrescine transaminase and polyamine oxidase, and quantifies putrescine by an enzyme cycling reaction using putrescine transaminase in the reaction in the reverse direction from putrescine to 4-aminobutanal. Specifically, hydrogen peroxide produced by the polyamine oxidase reaction is measured by a known color development method.
- a method using a dehydrogenase is known (for example, see Patent Documents 5 and 6).
- oxidized coenzyme NAD (P) or an analog thereof and reduced coenzyme NAD (P) or an analog thereof an enzyme cycling reaction utilizing the reversible reactivity of dehydrogenase is allowed to proceed, and a substrate for dehydrogenase is obtained.
- Quantitative determination with high sensitivity such as quantification of bile acids using 3 ⁇ -hydroxysteroid dehydrogenase in the presence of thio-NAD and reduced NAD, and quantification of glucose-6-phosphate using glucose-6-phosphate dehydrogenase Has been applied.
- a method for measuring triglyceride for example, see Non-Patent Document 2.
- a method for measuring creatinine is known (for example, see Patent Document 7).
- creatinine is converted into creatine by the action of creatinine amide hydrolase, and further converted into creatine phosphate and adenosine diphosphate (ADP) with creatine kinase in the presence of ATP, and then ADP is measured. Is a method for measuring creatinine.
- JP 2006-223163 A Japanese Patent Laid-Open No. 3-180200 Japanese Unexamined Patent Publication No. 7-155199 JP-A-7-177898 JP-A-3-224498 JP-A-4-335898 Japanese Patent Laid-Open No. 9-285297
- An object of the present invention is to provide a method and a composition capable of measuring with high accuracy at least one of a kinase positive reaction substrate, its phosphate, and a pre-induction substance induced to them.
- the measurement method using the enzyme cycling method is a technique that can be applied to high-sensitivity quantification by controlling the amount of enzyme because the signal due to the enzyme reaction increases according to the amount of enzyme added.
- a method using a kinase in an enzyme cycling method for quantifying a substrate has not been known so far, and measurement targets in the enzyme cycling method for quantifying a substrate have been limited.
- the normal reaction generally proceeds in preference to the reverse reaction in the kinase reaction, and it was considered technically difficult to apply the kinase to the enzyme cycling method using the reverse reaction.
- a method for measuring a substrate using a kinase includes a method for measuring creatinine using creatine kinase (see, for example, Patent Document 7). It has only been used for the amphatic conversion reaction. Therefore, when the concentration of the measurement object is low, sufficient sensitivity cannot always be obtained, and it has been necessary to deal with it by increasing the amount of the sample. However, there was a problem in terms of practicality, such as increasing the amount of specimens, making it more susceptible to coexisting substances.
- the present inventors utilize a coenzyme that catalyzes both the forward reaction and reverse reaction of the kinase reaction and has a different nucleoside moiety in the forward reaction and reverse reaction.
- the kinase positive reaction substrate and / or its phosphate, or the pre-induction substance induced to them can be measured with high sensitivity.
- the present invention has been completed.
- the present invention has the following configuration.
- [1] A method for measuring at least one of a positive reaction substrate of a kinase, a phosphoric acid oxide thereof, and a pre-induction substance induced therein, (1) A kinase that catalyzes a forward reaction and a reverse reaction in which a phosphate is produced from a forward reaction substrate of the kinase in the presence of a nucleotide coenzyme.
- a sample utilizing a nucleotide coenzyme having a nucleoside moiety, a first nucleotide coenzyme of the kinase, and a second nucleotide coenzyme having a different nucleoside moiety from the first nucleotide coenzyme Is a pre-induction substance, it is brought into contact with a sample that has been subjected to a quantitative induction of the pre-induction substance to the kinase's positive reaction substrate and / or its phosphate, and the cycling reaction of the following formula (1) is carried out.
- [1-1-1] The measurement method according to [1] or [1-1] above, wherein the measurement target is a kinase positive reaction substrate and / or a phosphoric acid oxide thereof, and the combination of the measurement target and the kinase is any one of those shown in Table 1. .
- the measurement target is a kinase positive reaction substrate and / or a phosphate thereof, and the combination of the measurement target and the kinase is any of the following, [1] to [1-1-1] Measuring method. In the following, it is described in the order of measurement object: kinase.
- the phosphoric acid oxide is a phosphoric acid oxide corresponding to the described kinase positive reaction substrate.
- the measurement target is a pre-induction substance
- the sample is a sample that has been subjected to a quantitative induction of the pre-induction substance to the positive reaction substrate of kinase and / or its phosphate, and the detected signal changes
- the measurement target is a pre-induction substance, and the combination of the measurement target, kinase, and the positive reaction substrate of kinase and / or its phosphorous oxide is one of those described in Table 2, above [1] or [1-2 ] The measuring method of description.
- the measurement target is a pre-induction substance
- the measurement target, the kinase, and the kinase positive reaction substrate / phosphorus oxide combination are any one of the following combinations, [1], [1-2] and [ 1-2-1].
- kinase kinase positive reaction substrate.
- Creatinine Creatine kinase (EC 2.7.3.2): Creatine / phosphate thereof Glyceraldehyde-3-phosphate, hydroxyacetone phosphate, glycerol-3-phosphate, lysophosphatidic acid, lysophosphatidylcholine, fructose- 1,6-bisphosphoric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, or 2,3-bisphosphoglyceric acid: 3-phosphoglycerate kinase (EC 2.7.2.3): 3-phosphoglyceric acid / Phosphoric acid glucose-6-phosphate: phosphofructo-1-kinase (EC 2.7.1.11): phosphofructose-6-phosphate / phosphoric acid droxyacetone phosphate, triglyceride, lysophosphatidylcholine Lysophosphatidic acid, glycerol-3-phosphate Lysophosphatidylgly
- the above-mentioned step (2) is a step of detecting a change amount of a signal corresponding to an increase amount of a positive reaction change product of the first nucleotide coenzyme or a reverse reaction change product of the second nucleotide coenzyme. 1] to [1-2-4].
- the nucleoside part of the first nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine.
- the method according to any one of [1] to [2] above, wherein the nucleoside part of the first nucleotide coenzyme and the second nucleotide coenzyme are different.
- nucleoside part of the first nucleotide coenzyme is adenosine, inosine, guanosine, or deoxyadenosine, and the nucleoside part of the first nucleotide coenzyme is different from that of the second nucleotide coenzyme.
- the nucleoside portion of the second nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine.
- nucleoside part of the second nucleotide coenzyme is adenosine, inosine, guanosine, or deoxyguanosine
- nucleoside part of the first nucleotide coenzyme and the second nucleotide coenzyme are different.
- the nucleoside portion of the first nucleotide coenzyme is adenosine, inosine, guanosine, or deoxyadenosine
- the nucleoside portion of the second nucleotide coenzyme is adenosine, inosine, guanosine, or deoxyguanosine
- nucleoside part of the first nucleotide coenzyme and the nucleoside part of the second nucleotide coenzyme is adenosine and inosine, guanosine and adenosine, deoxyadenosine and guanosine, deoxyadenosine and deoxyguanosine, deoxyadenosine and inosine, or inosine.
- the measurement method according to any one of [1] to [7] above, which is adenosine.
- nucleoside moiety of the first nucleotide coenzyme is adenosine and the nucleoside moiety of the second nucleotide coenzyme is inosine.
- ATP adenosine triphosphate
- IDDP inosine diphosphate
- adenosine diphosphate (ADP) -dependent glucokinase (EC 2.7.1.147) is used to increase the amount of adenosine diphosphate (ADP) in the presence of glucose.
- ADP adenosine diphosphate
- the measurement method according to any one of [1] to [8], wherein a change amount of a signal that changes corresponding to is detected.
- adenosine diphosphate (ADP) -dependent glucokinase (EC 2.7.1.147) is used to obtain thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotine.
- ADP adenosine diphosphate
- thioNADP thionicotinamide adenine dinucleotide phosphate
- thioNAD amidoadenine dinucleotide
- NADP nicotinamide adenine dinucleotide phosphate
- NAD nicotinamide adenine dinucleotide
- glucose glucose 6-phosphate dehydrogenase
- the measurement method according to any one of [1] and [1-2] to [10], wherein the measurement target is a method for measuring creatinine, which is a pre-induction substance, and includes the following steps.
- B) ATP C
- IDP Using adenosine diphosphate-dependent glucokinase (EC 2.7.1.147), any one of thio-NADP, thio-NAD, NADP, or NAD coenzyme, glucose, and glucose 6-phosphate Detecting a change in signal corresponding to an increased amount of adenosine diphosphate in the presence of dehydrogenase;
- a measurement composition for measuring at least one of a kinase positive reaction substrate, a phosphoric acid oxide thereof, and a pre-induction substance induced therein, and when the measurement target is a pre-induction substance, induction (A) a kinase, in the presence of a nucleotide coenzyme, used in the presence of a nucleotide coenzyme; A kinase that catalyzes a forward reaction to produce its phosphorous oxide from the reverse reaction and a reverse reaction thereof, and uses a nucleotide coenzyme having at least different nucleoside moieties in the forward reaction and the reverse reaction, (B) a first nucleotide coenzyme in a positive reaction; (C) a second nucleotide coenzyme in a reverse reaction, wherein the nucleoside moiety is different from the first nucleotide coenzyme; A composition comprising:
- [12-1-1] The composition according to [12] or [12-1] above, wherein the measurement target is a positive reaction substrate of kinase and / or a phosphate thereof, and the combination of the measurement target and kinase is any one of those shown in Table 3. object.
- measurement target is a pre-induction substance
- the combination of the measurement target, kinase, and kinase positive reaction substrate / phosphorus oxide thereof is one of the following combinations: 12-2-1].
- measurement target kinase: kinase positive reaction substrate / phosphorus oxide thereof are described in this order.
- Creatinine Creatine kinase (EC 2.7.3.2): Creatine / phosphate thereof Glyceraldehyde-3-phosphate, hydroxyacetone phosphate, glycerol-3-phosphate, lysophosphatidic acid, lysophosphatidylcholine, fructose- 1,6-bisphosphoric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, or 2,3-bisphosphoglyceric acid: 3-phosphoglycerate kinase (EC 2.7.2.3): 3-phosphoglyceric acid / Phosphoric acid glucose-6-phosphate: phosphofructo-1-kinase (EC 2.7.1.11): phosphofructose-6-phosphate / phosphoric acid thereof), (dihydroxyacetone phosphate, triglyceride, Lysophosphatidylcholine, lysophosphatidic acid, glycerol -Phosphate, lys
- the measurement target is creatinine which is a pre-induction substance
- the kinase is creatine kinase
- the positive reaction substrate of the kinase is creatine, according to any one of [12] and [12-2] to [12-2-2] above Composition.
- the nucleoside portion of the first nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine.
- nucleoside part of the first nucleotide coenzyme is adenosine, inosine, guanosine, or deoxyadenosine, and the nucleoside part of the first nucleotide coenzyme and the second nucleotide coenzyme are different.
- nucleoside part of the first nucleotide coenzyme and the second nucleotide coenzyme are different.
- the nucleoside portion of the second nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine.
- nucleoside part of the second nucleotide coenzyme is adenosine, inosine, guanosine, or deoxyguanosine, and the nucleoside part of the first nucleotide coenzyme and the second nucleotide coenzyme are different.
- nucleoside part of the first nucleotide coenzyme and the second nucleotide coenzyme are different.
- the nucleoside portion of the first nucleotide coenzyme is adenosine, inosine, guanosine, or deoxyadenosine
- the nucleoside portion of the second nucleotide coenzyme is inosine, guanosine, adenosine, or deoxyguanosine
- nucleoside part of the first nucleotide coenzyme and the nucleoside part of the second nucleotide coenzyme is adenosine and inosine, guanosine and adenosine, deoxyadenosine and guanosine, deoxyadenosine and deoxyguanosine, deoxyadenosine and inosine, or guanosine
- composition according to any one of [12] to [17], which is adenosine.
- nucleoside part of the first nucleotide coenzyme is adenosine and the nucleoside part of the second nucleotide coenzyme is inosine.
- the first nucleotide coenzyme is adenosine triphosphate (ATP), and the second nucleotide coenzyme is inosine diphosphate (IDP), according to any one of [12] to [17-2] above Composition.
- ATP adenosine triphosphate
- IDP inosine diphosphate
- a change in the first nucleotide coenzyme by the forward reaction can be used, but the detection enzyme that cannot use the second nucleotide coenzyme or the first nucleotide coenzyme cannot be used, but the second nucleotide complement by the reverse reaction can be used.
- ADP adenosine diphosphate
- EC 1.7.1.147 adenosine diphosphate
- thioNADP Thionicotinamide adenine dinucleotide phosphate
- thioNAD thionicotinamide adenine dinucleotide
- NADP nicotinamide adenine dinucleotide phosphate
- NAD nicotinamide adenine dinucleotide
- Glucose 6-phosphate dehydrogenase The composition according to [18], further comprising:
- the measurement target is creatinine, a pre-induction substance,
- the creatinine is used after being contacted with creatinine amide hydrolase (EC 3.5.2.10) in the presence of water and quantitatively inducing creatinine into creatine,
- A creatine kinase
- B adenosine triphosphate (ATP);
- C inosine diphosphate (IDP);
- D adenosine diphosphate (ADP) dependent glucokinase (EC 2.7.1.147);
- E glucose;
- G glucose 6-phosphate dehydrogenase;
- the composition according to any one of the following
- a reagent kit comprising the composition according to any one of [12] to [21].
- a kinase substrate it is possible to measure at least one of a kinase substrate, its phosphate, and a pre-induction substance induced to them with high sensitivity.
- 2 is a graph showing the correlation between fructose-6-phosphate concentration in a sample using phosphofructo-1-kinase and the amount of change in absorbance. It is a graph which shows the correlation with the glycerol density
- 3 is a graph showing the correlation between the concentration of 3-phosphoglycerate in a sample using 3-phosphoglycerate kinase and the amount of change in absorbance.
- 2 is a graph showing the correlation between the glucose-6-phosphate concentration in a sample using hexokinase and the amount of change in absorbance. It is a graph which shows the correlation with the glucose-6-phosphate density
- ADP adenosine diphosphate
- the method according to the present embodiment is a method for measuring at least one of a positive reaction substrate of a kinase, a phosphoric acid oxide thereof, and a pre-induction substance induced therein, (1) A kinase that catalyzes a forward reaction and a reverse reaction in which a phosphate is produced from a forward reaction substrate of the kinase in the presence of a nucleotide coenzyme.
- the pre-induction substance is brought into contact with a sample that has been subjected to a quantitative induction of the pre-induction substance to a kinase reaction substrate and / or its phosphate, and enzyme cycling of the following formula (3) Reacting the reaction; and (2) Change in signal corresponding to the amount of change in at least one of the first nucleotide coenzyme, the first nucleotide coenzyme change, the second nucleotide coenzyme, and the second nucleotide coenzyme change Detecting the amount; (3) calculating the amount of at least one of the positive reaction substrate of the kinase contained in the sample
- the kinase used in the method according to the present embodiment catalyzes a positive reaction that generates its phosphate from a positive reaction substrate in the presence of the first nucleotide coenzyme, In addition, in the presence of the second nucleotide coenzyme, the kinase is capable of catalyzing a reverse reaction that generates a positive reaction substrate from a phosphate of the positive reaction substrate.
- the measurement target is a pre-induction substance
- a sample subjected to a treatment for quantitatively inducing the pre-induction substance to the positive reaction substrate of kinase and / or its phosphorous oxide is used.
- the positive reaction substrate of the kinase to be measured by the method according to the present embodiment and its phosphate as long as it can be measured by the method according to the present embodiment.
- creatine, 3-phosphoglycerin Examples include acids, pyruvic acid, fructose-6-phosphate, glycerol, and their phosphorus oxides.
- Examples of the pre-induction substance induced therein include creatinine, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, glycerol-3-phosphate, lysophosphatidic acid, lysophosphatidylcholine, fructose-1,6-bisphosphate, Examples include 2-phosphoglycerate, phosphoenolpyruvate, 2,3-bisphosphoglycerate, glucose-6-phosphate, triglyceride, phosphatidylcholine, phosphatidylglycerol, lysophosphatidylglycerol, and glucose-1-phosphate. However, it is not limited to these. For example, a preferable pre-induction substance is creatinine.
- the sample according to the present embodiment is not particularly limited as a sample to be examined for the amount of the positive reaction substrate of kinase and / or its phosphate, or the pre-induction substance induced therein, for example, human Alternatively, it is an animal biological sample, preferably human blood, more preferably human serum. Whether or not the sample contains the positive reaction substrate of the kinase to be measured and / or its phosphate, or the pre-induction substance induced therein, was unknown before the above method was performed. It is possible to perform the method according to the present embodiment, and it becomes clear that the sample does not contain the kinase's positive reaction substrate and / or its phosphate, or the pre-induction substance induced therein. It can happen.
- the “pre-induction substance” is a substance that is induced to a positive reaction substrate of kinase and / or a phosphoric acid oxide thereof.
- the pre-induction substance includes all of the starting substance and intermediate substance, and may be any of them.
- the “quantitative induction treatment” means a decrease amount of a substance before induction and an increase amount of a positive reaction substrate of kinase which is a substance after induction and / or its phosphate.
- it refers to a treatment in which a pre-induction substance is induced to the positive reaction substrate of kinase and / or its phosphate.
- Any processing that can calculate the amount of the pre-induction substance based on the amount of change in the signal corresponding to the amount of change may be used.
- a known chemical treatment may be used as an example of the quantitative induction process, but is not limited thereto.
- Examples of chemical treatment include, but are not limited to, treatments using chemical reactions such as hydrolysis, oxidation, and reduction. Specific examples include hydrolysis with acid or alkali.
- Examples other than chemical treatment include enzymatic treatment.
- creatinine can be induced into creatine by contacting with creatinine amide hydrolase (creatininase) (EC 3.5.2.10) in the presence of water. Therefore, in the method according to the present embodiment, if creatine is measured by using creatine kinase as a kinase, creatinine, which is a pre-induction substance for creatine, can be measured.
- triglyceride can be induced to glycerol by lipase (EC 3.1.1.3), triglyceride can be measured by combining with an enzyme cycling reaction of glycerokinase.
- lysolecithin is induced into choline and glycerol-3-phosphate by lysophospholipase (EC 3.1.1.5) and glycerophosphocholine phosphodiesterase (EC 3.1.4.2). Therefore, lysolecithin can also be measured by measuring glycerol-3-phosphate by causing an enzyme cycling reaction of glycerokinase using induced glycerol-3-phosphate as a substrate.
- the enzymatic treatment is not limited to these.
- the kinase that can be used in the method according to the present embodiment can catalyze the forward reaction and its reverse reaction in the presence of a nucleotide coenzyme to produce its phosphate from the forward reaction substrate of the kinase.
- the “kinase positive reaction” in the method according to the present embodiment refers to a reaction in a direction in which the phosphate is transferred from the nucleotide coenzyme to the positive reaction substrate among the high energy phosphate transfer catalyzed by the kinase.
- the number of phosphoric acids to be transferred does not matter.
- the number of transferred phosphates is one.
- the “kinase reverse reaction” refers to a reaction in the reverse direction to the “kinase normal reaction”.
- the measurement target is a kinase reaction substrate and / or its phosphate
- examples of the combination of the measurement target and the kinase include, but are not limited to, the following.
- the measurement object is described in the order of kinase.
- the phosphoric acid oxide is a phosphoric acid oxide corresponding to the positive reaction substrate of the described kinase.
- Creatine and / or its phosphate creatine kinase 3-phosphoglycerate and / or its phosphate: 3-phosphoglycerate kinase Pyruvate and / or its phosphate: pyruvate kinase fructose-6-phosphate and / Or its phosphate: phosphofructo-1-kinase glycerol and / or its phosphate: glycerol kinase hexose and / or its phosphate: hexokinase glucose and / or its phosphate: adenosine diphosphate (ADP) dependent Sex glucokinase
- ADP adenosine diphosphate
- Preferable examples include, but are not limited to, the following combinations including kinases specified by EC number (Enzyme Commission numbers). Creatine and / or its phosphate: creatine kinase (EC 2.7.3.2) 3-phosphoglycerate and / or its phosphate: 3-phosphoglycerate kinase (EC 2.7.2.3) Pyruvate and / or its phosphate: pyruvate kinase (EC 2.7.1.40) Fructose-6-phosphate and / or its phosphate: phosphofructo-1-kinase (EC 2.7.1.11) Glycerol and / or its phosphate: glycerolase (EC 2.7.1.30) Hexose and / or its phosphate: hexokinase (EC 2.7.1.1) Glucose and / or its phosphate: ADP-dependent glucokinase (EC 2.7.1.1147)
- the measurement target is a pre-induction substance
- examples of the combination of the measurement target, the kinase, and the positive reaction substrate of the kinase and / or the phosphoric acid thereof include the following, but are not limited thereto.
- Creatinine Creatine kinase: Creatine and / or its phosphate glyceraldehyde-3-phosphate, hydroxyacetone phosphate, glycerol-3-phosphate, lysophosphatidic acid, lysophosphatidylcholine, fructose-1,6-bisphosphate, 2 -Phosphoglyceric acid, phosphoenolpyruvate, or 2,3-bisphosphoglyceric acid: 3-phosphoglycerate kinase: 3-phosphoglyceric acid and / or its phosphate glucose-6-phosphate: phosphofructo-1- Kinase: Phosphofructose-6-phosphate and / or its phosphate dihydroxyacetone phosphate, triglyceride, lysophosphatidylcholine, lysophosphatidic acid, glycerol-3-phosphate, lysophosphatidylglycerol, or phosphine
- Creatinine Creatine kinase (EC 2.7.3.2): Creatine and / or its phosphate glyceraldehyde-3-phosphate, hydroxyacetone phosphate, glycerol-3-phosphate, lysophosphatidic acid, lysophosphatidylcholine, Fructose-1,6-bisphosphate, 2-phosphoglycerate, phosphoenolpyruvate, or 2,3-bisphosphoglycerate: 3-phosphoglycerate kinase (EC 2.7.2.3): 3-phospho Glyceric acid and / or its phosphate
- Glucose-6-phosphate Phosphofructo-1-kinase (EC 2.7.1.11): Phosphofructose-6-phosphate and / or its phosphate Droxyacetone phosphorus Acid, triglyceride,
- Creatinine Creatine kinase: Creatine and / or its phosphate
- the measurement target is a kinase positive reaction substrate and / or its phosphate
- the measurement target of the method according to the present embodiment and the combination of each of the kinases are determined by the following method in addition to the above. be able to.
- the combination of the measurement target, kinase, and the positive reaction substrate of the kinase / phosphorus oxide thereof can be determined by the following method in addition to the above. .
- a specific kinase is selected with reference to the enzyme handbook (Asakura Shoten, 1982), for example. The selection is made with reference to whether or not the reverse reaction proceeds or literature information on the specificity to the nucleotide coenzyme can be obtained. Moreover, when literature information is not obtained, such information can actually be confirmed if it combines with methods, such as a well-known high performance liquid chromatography (HPLC) method.
- HPLC high performance liquid chromatography
- glycerol or adenosine triphosphate may be detected by an existing method.
- glycerol when glycerol is detected, it can be detected as hydrogen peroxide using glycerol oxidase.
- ATP adenosine triphosphate
- the reverse reaction When the reverse reaction is confirmed, if the pH during the reaction is further changed and the optimum pH for the reverse reaction is examined, it can be used as a reference when setting the execution conditions of the method according to the present embodiment. .
- the specificity of the forward and / or reverse reaction nucleotide coenzyme can be confirmed by combining with the HPLC method.
- the kinase used in the method according to this embodiment has a specificity of 4% or more, preferably 8% or more, for a nucleotide coenzyme having a different nucleoside moiety that works best for each of the forward reaction and the reverse reaction.
- the present invention is not limited to this.
- a positive reaction substrate or a phosphor oxide thereof to be combined with these kinases is selected.
- the selection of the positive reaction substrate or its phosphate may be selected by the Michaelis constant Km of the kinase for the positive reaction substrate or its phosphate.
- the Michaelis constant Km is preferably as small as possible.
- the Km value is 100 mmol / L, preferably 50 mmol / L or less, but is not limited thereto.
- these pre-induction substances can be measured. That is, these pre-induction substances can be converted to a kinase reaction substrate and / or a phosphoric acid oxide thereof by a known chemical treatment. Furthermore, confirm the metabolic pathway that is converted to the kinase's positive reaction substrate and / or its phosphate with a metabolic map, etc., and if the enzymes involved in the reaction are readily available, these pre-induction substances are to be measured. It can also be.
- nucleotide coenzyme refers to a nucleoside phosphate that acts as a kinase coenzyme.
- the first nucleotide coenzyme and the second nucleotide coenzyme used in the method according to the present embodiment are nucleotide coenzymes having different nucleoside moieties from each other.
- the type of the nucleoside part of the nucleotide coenzyme used in the method according to the present embodiment is not particularly limited, but examples include adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, Examples include deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine.
- these phosphorus oxides may be monophosphorus oxide, 2-phosphorus oxide, or 3-phosphorus oxide.
- nucleotide coenzymes examples include ribonucleotides and deoxyribonucleotides.
- ribonucleotides as nucleotide coenzymes include ATP, ADP, adenylate (AMP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanylate (GMP), 5-methyluridine triphosphate Acid (TTP), 5-methyluridine diphosphate (TDP), 5-methyluridine monophosphate (TMP), uridine triphosphate (UTP), uridine diphosphate (UDP), uridine monophosphate (UMP) , Cytidine triphosphate (CTP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), xanthosine triphosphate (XTP), xanthosine diphosphate (XDP), xanthosine monophosphate (XMP), inosine Examples thereof include, but are not limited to, triphosphate (ITP), inosine diphosphate (IDP), and inosine monophosphate (IMP).
- AMP guanosine triphosphat
- deoxyribonucleotides as nucleotide coenzymes include deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), deoxyguanosine triphosphate (dGTP) , Deoxyguanosine diphosphate (dGDP), deoxyguanosine monophosphate (dGMP), thymidine triphosphate (dTTP), thymidine diphosphate (dTDP), thymidine monophosphate (dTMP), deoxyuridine triphosphate (dUTP) ), Deoxyuridine diphosphate (dUDP), deoxyuridine monophosphate (dUMP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP), deoxycytidine monophosphate (dCMP), deoxyxanthosine 3-phosphate (dXTP), deoxyxa Tonsine 2 phosphate (dATP),
- Preferred nucleotide coenzymes are, for example, ATP, ADP, GTP, GDP, TTP, TDP, UTP, UDP, CTP, CDP, XTP, XDP, ITP, IDP, dATP, dADP, dGTP, dGDP, dTTP, dTDP, dUTP, Examples include, but are not limited to, dUDP, dCTP, dCDP, dXTP, dXDP, dITP, and dIDP.
- a substituent having a coloring ability may be bound to these nucleotide enzymes.
- the nucleoside portion of the first nucleotide coenzyme in the positive reaction used in the method according to the present embodiment is not particularly limited, but examples include adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxy. Examples include guanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine.
- adenosine, inosine, guanosine, deoxyadenosine and the like can be mentioned. More preferably, adenosine is mentioned.
- these phosphorous oxides may be monophosphoric oxides, two phosphorous oxides or three phosphorous oxides, but three phosphorous oxides are preferable.
- Specific examples of the first nucleotide coenzyme include ATP, GTP, TTP, UTP, CTP, XTP, ITP, dATP, dGTP, dTTP, dUTP, dCTP, dXTP, and dITP in addition to the specific examples described above. Etc.
- the nucleoside portion of the second nucleotide coenzyme used in the reverse reaction used in the method according to the present embodiment is not particularly limited, but examples include adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxy.
- examples include guanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine.
- Preferable examples include inosine, guanosine, adenosine, deoxyguanosine and the like. More preferred is inosine.
- these phosphorous oxides may be monophosphoric oxides, biphosphoric oxides, or triphosphorous oxides, but diphosphorous oxides are preferred.
- Specific examples of the second nucleotide coenzyme include ADP, GDP, TDP, UDP, CDP, XDP, IDP, dADP, dGDP, dTDP, dUDP, dCDP, dXDP, and dIDP in addition to the specific examples described above. Is mentioned.
- the preferred nucleoside moiety of the first nucleotid coenzyme and the second nucleotide coenzyme used in the method according to the present embodiment is not particularly limited, but is, for example, adenosine, inosine, guanosine, deoxyadenosine, etc. And they are different from each other.
- the combination of the nucleoside portion of the first nucleotide coenzyme and the nucleoside portion of the second nucleotide coenzyme is adenosine and inosine, guanosine and adenosine, deoxyadenosine and guanosine, deoxyadenosine and deoxyguanosine, deoxyadenosine and inosine, or Inosine and adenosine.
- the nucleoside portion of the first nucleotide coenzyme is adenosine and the nucleoside portion of the second nucleotide coenzyme is inosine.
- the “change in the first nucleotide coenzyme due to the positive reaction” refers to the transfer of the phosphate from the first nucleotide coenzyme to the positive reaction substrate of the kinase in the kinase positive reaction. It refers to the dephosphorylating oxide of the first nucleotide coenzyme later.
- the “change in the second nucleotide coenzyme due to the reverse reaction” refers to the second nucleotide after the transfer of phosphate from the kinase reverse reaction substrate to the second nucleotide coenzyme in the kinase reverse reaction. This refers to the phosphorylate of coenzyme.
- the number of transferred phosphoric acid may be one, two, or three. Preferably one.
- the cycling reaction of the above formula (3) is performed, for example, in the following manner.
- an excess amount of the first and second nucleotide coenzymes specifically 0.1 mmol / L or more and 12 mmol / L or less, more preferably 0.2 mmol / L to 6 mmol / L is prepared, and the pH during the reaction is prepared.
- a pH buffer is added at 20 mmol / L or more and 200 mmol / L or less to obtain a reaction solution.
- the pH at the time of the reaction may be appropriately selected under conditions that allow the reaction to proceed efficiently.
- the pH is adjusted to near neutrality between pH 5.5 and pH 8.5, and a metal such as magnesium chloride, which is an activator of kinase.
- a salt is added in a range of 0.5 mmol / L to 10 mmol / L.
- the aforementioned kinase is added thereto, and the reaction solution is preliminarily heated to 25 ° C. or higher and 42 ° C. or lower, more preferably around 37 ° C., and a small amount of positive reaction substrate and What is necessary is just to add the phosphorous oxide and / or start reaction.
- the amount of enzyme added will be described below.
- the cycling rate depends on the amount of enzyme, and it is known that the smaller the Michaelis constant (Km value) for the substrate, the higher the sensitivity can be obtained with a small amount of enzyme. Therefore, as a measure of the amount of enzyme to be added, the enzyme unit (unit: more than 1 time, preferably 2 times or more of the larger Km value (mmol / L) of the positive reaction substrate and its phosphoric oxide per 1 mL of the reaction solution)
- the upper limit of u may be added in an amount that can be substantially added.
- the Km value can also be obtained by a known method. These amounts are merely examples and are not limited to these.
- CK creatine kinase
- CK is an enzyme widely present in the animal kingdom and is abundantly contained in skeletal muscle, myocardium, brain and the like.
- CKs originating from rabbits, cattle, pigs, chickens, etc. are commercially available, and information on the specificity of nucleotide coenzymes can be obtained from the literature.
- IDP has been reported to show a relative activity of 29% when ADP is 100% (J Biol Chem., 241, 3116-3125, 1966).
- creatine amide hydrolase EC 3.5.3.3.
- sarcosine oxidase EC
- CK sarcosine oxidase
- the specificity of CK for nucleoside diphosphates other than ADP can be easily confirmed by generating hydrogen peroxide using EC 1.5.3.1) and quantifying the hydrogen peroxide.
- ATP can be selected as the first nucleotide coenzyme in the forward reaction and IDP can be selected as the second nucleotide in the reverse reaction.
- a pH at which the cycling reaction efficiently proceeds may be appropriately selected.
- a neutral buffer solution of pH 6 or more and pH 8 or less may be optimal.
- An excess amount of ADP and IDP, more specifically 0.1 mmol / L or more and 12 mmol / L or less, preferably 0.2 mmol / L or more and 6 mmol / L or less is present, and a small amount of creatine or ADP and IDP or What is necessary is just to add creatine phosphate and enzyme CK.
- the Km values for creatine and creatine phosphate are reported to be 16 mmol / L and 5 mmol / L, respectively (J Biol Chem. 210, p65, 1954). According to the above-mentioned enzyme addition amount guideline, these correspond to 16 u / mL and 32 u / mL. Therefore, the enzyme is preferably added at 32 u / mL or more, and the upper limit is about 800 u / mL, but is not limited thereto.
- step (2) of the method according to the present embodiment the first nucleotide coenzyme, the change in the first nucleotide coenzyme by the forward reaction, the second nucleotide coenzyme, and the second nucleotide by the reverse reaction
- change_quantity of the signal corresponding to either of the variation
- the amount of change in the signal may be the amount of decrease in the first nucleotide coenzyme or the second nucleotide coenzyme, or the change in the first nucleotide coenzyme due to the forward reaction or the change in the second nucleotide coenzyme due to the reverse reaction.
- it is an increased amount of a change in the first nucleotide coenzyme due to the forward reaction or a change in the second nucleotide coenzyme due to the reverse reaction.
- a known HPLC method or the like can be used as a method for detecting the amount of change.
- the HPLC method add a chelating agent or the like to complete the enzyme reaction, and then change the first nucleotide change by the forward reaction or the second nucleotide coenzyme change by the reverse reaction, such as a reverse phase column, as appropriate. Select and quantify. Information on these HPLC analysis methods is readily available from resin manufacturers.
- a change in the first nucleotide coenzyme due to a positive reaction can be used, but a detection enzyme that cannot use the second nucleotide coenzyme is used, You may detect the signal variation
- a detection enzyme that cannot use the first nucleotide coenzyme but can use the change in the second nucleotide coenzyme due to the reverse reaction increase the amount of the change in the second nucleotide coenzyme due to the reverse reaction.
- the amount of signal change that changes may be detected.
- ADP-dependent glucokinase adenosine diphosphate-dependent glucokinase (ADP-dependent glucokinase, EC 2.7.1.147)
- ADP-dependent glucokinase derived from Pyrococcus furiosus can use CDP in addition to ADP as a nucleotide coenzyme, but is known to have very little effect on GDP and IDP.
- ADP-dependent glucokinase coexists as an enzyme for detection during the kinase cycling reaction using ATP as the first nucleotide coenzyme and IDP or GDP as the second nucleotide coenzyme
- the first reaction by the positive reaction is performed. Only ADP, which is a variation of the nucleotide coenzyme, can be specifically detected. This has been actually confirmed as will be described later.
- glycerol kinase derived from E. coli uses only ATP as a phosphate donor. Therefore, for example, when a kinase using ITP, GTP or CTP as the first nucleotide coenzyme and ADP as the second nucleotide coenzyme is used in the method according to this embodiment, glycerol kinase derived from E. coli is used as the detection enzyme. If used, only ATP can be specifically detected. As a detection enzyme that specifically detects only ATP, in addition to the above-mentioned glycerol kinase derived from Escherichia coli, other kinases specific to only ATP can be used.
- step (2) of the method according to the present embodiment paying attention to the combination of the first nucleotide coenzyme and the second nucleotide coenzyme of the kinase used in the method according to the present embodiment, What is necessary is just to select the detection enzyme which can be utilized suitably.
- the specificity for the coenzyme may depend not only on the enzyme origin but also on conditions such as the amount of coenzyme and the amount of enzyme added. Therefore, there are cases where the results are different from those reported in the literature, and it is better to actually confirm the specificity when selecting.
- adenosine diphosphate-dependent glucokinase (EC 2.7.1.147) is used as the detection enzyme.
- the product AMP or glucose 6-phosphate can be quantified by a known method.
- Glucose-6-phosphate corresponds to increased amounts of ADP in the presence of thio-NADP, thio-NAD, NADP, and NAD coenzymes, glucose, and glucose 6-phosphate dehydrogenase, as the case may be. In some cases, the amount of change in the signal that changes in response to the change is detected.
- glucose 6-phosphate can be measured as a change in absorbance at 340 nm based on reduced NAD (P) using glucose 6-phosphate dehydrogenase (G6PDH) in the presence of NAD (P).
- G6PDH glucose 6-phosphate dehydrogenase
- P thio-NAD
- P thio-NAD
- nitrotetrazolium blue (Nitro-NB)
- 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, sodium salt (WST-1)
- WST- 2-methoxyPMS which is an electron carrier in the presence of a hydrogen acceptor such as 3
- absorbance in the visible region as a formazan dye with the enzyme diaphorase.
- glucose 6-phosphate can be quantified with high sensitivity by an enzyme cycling method using G6PDH (see, for example, Patent Document 7).
- the step of detecting the change amount of these changing signals may be performed simultaneously with the cycling reaction in the method according to the present embodiment, or may be performed separately.
- the product glycerol-3-phosphate can be quantified by a known method.
- the method according to the present embodiment can be carried out with one kind of kinase, but the cycling reaction can also be carried out using two or more kinds of enzymes that catalyze the same reaction with different origins.
- a known method is used as a method for calculating the amount of the positive reaction substrate of kinase and / or its phosphate, or the pre-induction substance induced in the sample in the sample.
- Can do for example, in the enzyme cycling reaction, the change in the first nucleotide coenzyme due to the forward reaction and the change in the second nucleotide coenzyme due to the reverse reaction tend to increase in proportion to time.
- To calculate the amount of a test substance in a sample specify the reaction time of the enzyme cycling reaction (for example, 5 to 7 minutes after the start of the reaction), and select a substance (calibrator) whose concentration is a known standard.
- the amount of analyte in the sample can be calculated by measuring the amount of change in signal corresponding to the calibrator variation.
- the calibrator can be a kinase reaction substrate or a phosphate thereof.
- the kinase's positive reaction substrate and / or the positive reaction substrate's phosphate After carrying out the step leading to the above, an enzyme cycling reaction may be carried out.
- an enzyme cycling reaction may be carried out.
- the step of leading the pre-induction substance to the kinase's positive reaction substrate and / or phosphoric acid of the positive reaction substrate is an enzyme reaction, if an excessive amount of an enzyme that catalyzes the induction reaction is added, the kinase's positive reaction substrate can be obtained in a short time.
- the cycling reaction of the method according to the present embodiment can be carried out by previously containing a kinase. In this case, what is necessary is just to prescribe
- the present embodiment is a composition for measuring at least one of a positive reaction substrate of kinase, its phosphate, and a pre-induction substance induced therein, (A) a kinase that catalyzes a forward reaction and a reverse reaction in which a phosphoric acid is produced from a forward reaction substrate of the kinase in the presence of a nucleotide coenzyme; A kinase utilizing a nucleotide coenzyme having a different nucleoside moiety; (B) a first nucleotide coenzyme in a positive reaction; (C) a second nucleotide coenzyme in a reverse reaction, wherein the nucleoside moiety is different from the first nucleotide coenzyme; A composition comprising: When the measurement target is a pre-induction substance, the composition is used after a treatment for quantitatively inducing the pre-induction substance to the kinase's positive reaction substrate
- the kinase of the composition according to the present embodiment can catalyze the forward reaction and the reverse reaction thereof in the presence of a nucleotide coenzyme to produce its phosphate from the kinase's forward reaction substrate.
- a nucleotide coenzyme having at least different nucleoside moieties can be used.
- the measurement target of the composition according to the present embodiment is a kinase reaction substrate and / or a phosphoric acid oxide thereof
- examples of the combination of the measurement target and the kinase are as follows. It is not limited. In addition, in the following illustration, it describes in order of a measuring object: kinase.
- the phosphoric acid oxide is a phosphoric acid oxide corresponding to the positive reaction substrate of the described kinase.
- Creatine and / or its phosphate creatine kinase 3-phosphoglycerate and / or its phosphate: 3-phosphoglycerate kinase Pyruvate and / or its phosphate: pyruvate kinase fructose-6-phosphate and / Or its phosphate: phosphofructo-1-kinase glycerol and / or its phosphate: glycerol kinase hexose and / or its phosphate: hexokinase glucose and / or its phosphate: ADP-dependent glucokinase
- Preferred examples include, but are not limited to, the following combinations including the following kinases identified by EC number. Creatine and / or its phosphate: creatine kinase (EC 2.7.3.2) 3-phosphoglycerate and / or its phosphate: 3-phosphoglycerate kinase (EC 2.7.2.3) Pyruvate and / or its phosphate: pyruvate kinase (EC 2.7.1.40) Fructose-6-phosphate and / or its phosphate: phosphofructo-1-kinase (EC 2.7.1.11) Glycerol and / or its phosphate: glycerolase (EC 2.7.1.30) Hexose and / or its phosphate: hexokinase (EC 2.7.1.1) Glucose and / or its phosphate: ADP-dependent glucokinase (EC 2.7.1.1147)
- the measurement target is a pre-induction substance
- examples of the measurement target, the kinase, and the positive reaction substrate of the kinase and / or the phosphoric oxide thereof include the following, but are not limited thereto.
- Creatinine Creatine kinase: Creatine and / or its phosphate glyceraldehyde-3-phosphate, hydroxyacetone phosphate, glycerol-3-phosphate, lysophosphatidic acid, lysophosphatidylcholine, fructose-1,6-bisphosphate, 2 -Phosphoglyceric acid, phosphoenolpyruvate, or 2,3-bisphosphoglyceric acid: 3-phosphoglycerate kinase: 3-phosphoglyceric acid and / or its phosphate glucose-6-phosphate: phosphofructo-1- Kinase: Phosphofructose-6-phosphate and / or its phosphate dihydroxyacetone phosphate, triglyceride, lysophosphatidylcholine, lysophosphatidic acid, glycerol-3-phosphate, lysophosphatidylglycerol, or phosphine
- Creatinine Creatine kinase (EC 2.7.3.2): Creatine and / or its phosphate glyceraldehyde-3-phosphate, hydroxyacetone phosphate, glycerol-3-phosphate, lysophosphatidic acid, lysophosphatidylcholine, Fructose-1,6-bisphosphate, 2-phosphoglycerate, phosphoenolpyruvate, or 2,3-bisphosphoglycerate: 3-phosphoglycerate kinase (EC 2.7.2.3): 3-phospho Glyceric acid and / or its phosphate
- Glucose-6-phosphate Phosphofructo-1-kinase (EC 2.7.1.11): Phosphofructose-6-phosphate / its phosphate), (droxyacetone Phosphoric acid, triglyceride
- the combination of the measurement target and each of the kinases is the same as the method according to the present embodiment. It can be determined by the same method as described above. Further, even when the measurement target is a pre-induction substance, the combination of the measurement target, the kinase, the positive reaction substrate of the kinase and / or its phosphoric acid, and the above description of the method according to the present embodiment It can be determined in a similar manner.
- the first nucleotide coenzyme and the second nucleotide coenzyme are nucleotide coenzymes having different nucleoside moieties.
- the type of nucleoside moiety is not particularly limited, but examples include adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine Is mentioned.
- these phosphorous oxides may be monophosphoric oxides, biphosphoric oxides, or triphosphorous oxides.
- nucleotide coenzymes include ATP, ADP, AMP, GTP, GDP, GMP, TTP, TDP, TMP, UTP, UDP, UMP, CTP, CDP, CMP, XTP, XDP, XMP, ITP, IDP, IMP, Examples include, but are not limited to, dATP, dADP, dAMP, dGTP, dGDP, dGMP, dTTP, dTDP, dTMP, dUTP, dUDP, dUMP, dCTP, dCDP, dCMP, dXTP, dXDP, dXMP, dITP, dIDP, and dIMP. Not.
- ATP ATP, ADP, GTP, GDP, TTP, TDP, UTP, UDP, CTP, CDP, XTP, XDP, ITP, IDP, dATP, dADP, dGTP, dGDP, dTTP, dTDP, dUTP, dUDP, dCTP, dCDP , DXTP, dXDP, dITP, and dIDP.
- a substituent having a coloring ability may be bound to these nucleotide enzymes.
- the nucleoside portion of the first nucleotide coenzyme in the positive reaction used in the composition according to the present embodiment is not particularly limited, but examples include adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxy. Examples include adenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine.
- adenosine, inosine, guanosine, deoxyadenosine, deoxyadenosine and the like can be mentioned. More preferably, adenosine is used.
- these phosphorus oxides may be monophosphorus oxide, 2-phosphorus oxide, or triphosphorus oxide, but triphosphorous oxide is preferable.
- Specific examples of the first nucleotide coenzyme include ATP, GTP, TTP, UTP, CTP, XTP, ITP, dATP, dGTP, dTTP, dUTP, dCTP, dXTP, and dITP in addition to the specific examples described above. Etc.
- the nucleoside portion of the second nucleotide coenzyme used in the composition according to the present embodiment in the reverse reaction is not particularly limited, but examples include adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine. , Deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, deoxyinosine and the like. Preferable examples include inosine, guanosine, adenosine, deoxyguanosine and the like. More preferred is inosine.
- these phosphorus oxides may be monophosphorus oxides, 2-phosphorus oxides, or 3-phosphorus oxides, but 2-phosphorus oxides are preferred.
- Specific examples of the second nucleotide coenzyme include ADP, GDP, TDP, UDP, CDP, XDP, IDP, dADP, dGDP, dTDP, dUDP, dCDP, dXDP, and dIDP in addition to the specific examples described above. Is mentioned.
- the combination of the nucleoside moieties of the first nucleotide coenzyme and the second nucleotide coenzyme in the composition of the present embodiment is not particularly limited as long as they are different from each other.
- adenosine and inosine, guanosine and adenosine Deoxyadenosine and guanosine, deoxyadenosine and deoxyguanosine, deoxyadenosine and inosine, or inosine and adenosine.
- the nucleoside portion of the first nucleotide coenzyme is adenosine and the nucleoside portion of the second nucleotide coenzyme is inosine.
- composition according to this embodiment is used, for example, the method according to this embodiment can be carried out, and the reaction of the above formula (3) can be caused to react by a cycling method.
- the first nucleotide coenzyme, the change in the first nucleotide coenzyme by the forward reaction, the second nucleotide coenzyme, and the second nucleotide coenzyme by the reverse reaction It is possible to detect a change amount of a signal corresponding to any one change amount of the change object. Moreover, even if the amount of change in the signal is the amount of decrease in the first nucleotide coenzyme or the second nucleotide coenzyme, the change in the first nucleotide coenzyme due to the forward reaction or the change in the second nucleotide coenzyme due to the reverse reaction It may be an increased amount of things.
- the amount of change in the signal is an increase in the change in the first nucleotide coenzyme due to the forward reaction or the change in the second nucleotide coenzyme due to the reverse reaction.
- a known HPLC method or the like can be used as a method for detecting the amount of change. The case of using the HPLC method is the same as the description of the method according to the present embodiment.
- composition according to the present embodiment it is possible to calculate the amount of the positive reaction substrate of kinase and / or its phosphate, or the pre-induction substance induced therein, contained in the sample. .
- the calculation method is the same as the description of the method according to the present embodiment, and a known method can be used.
- the composition according to the present embodiment can use a change in the first nucleotide coenzyme due to the positive reaction, but cannot use the detection enzyme that cannot use the second nucleotide coenzyme or the first nucleotide coenzyme.
- the detection enzyme include, but are not limited to, adenosine diphosphate-dependent glucokinase (EC 2.7.1.147).
- thio-NADP, thio-NAD, NADP or NAD coenzyme, glucose, and glucose 6-phosphate dehydrogenase may be added.
- the composition according to the present embodiment may be a reagent kit divided into a plurality of parts.
- the composition containing the second nucleotide coenzyme can be distributed into reagent kits that are appropriately divided into two or three or more.
- thio-NADP, thio-NAD, NADP or NAD, glucose, and a change in the first nucleotide coenzyme due to a positive reaction can be used, but the second nucleotide coenzyme cannot be used.
- the enzyme or the first nucleotide coenzyme cannot be used, but the second nucleotide coenzyme change due to the reverse reaction can be used.
- the composition containing the detection enzyme that can be used is appropriately distributed to the above two or more reagent kits. Can do. Any of these components can be included in duplicate reagent kits.
- the composition containing ATP, CK, and IDP can be appropriately divided into two reagent kits.
- NADP, ADP-dependent glucokinase, and glucose-6-phosphate dehydrogenase may be appropriately distributed into two reagent kits. Any of these components can be included in duplicate reagent kits.
- ADP-HKPII (T-92) Asahi Kasei Pharma Co., Ltd.) cannot use IDP and GDP as nucleoside-2-phosphate as a phosphate donor in addition to ADP. confirmed. That is, glucose produced by the ADP-HKPII reaction was measured as a change in absorbance at 340 nm based on reduced NADP at 37 ° C. in the presence of glucose-6-phosphate dehydrogenase (G6PDH: Toyobo) in the presence of NADP.
- G6PDH glucose-6-phosphate dehydrogenase
- a reaction solution having the following composition was prepared. 50 mmol / L Tris-HCl pH 8.0 10 mmol / L glucose 2 mmol / L magnesium chloride (MgCl 2 ) 1mmol / L NADP 1u / mL G6PDH 1 mmol / L nucleoside-2-phosphate (ADP, IDP, or GDP)
- the reagent blank was prepared by adding physiological saline instead of nucleoside-2-phosphate.
- Example 1 Confirmation of reversible reaction in the presence of ATP and IDP
- ATP is used as the first nucleotide coenzyme
- ATP is used as the second nucleotide coenzyme.
- IDP was selected as the nucleoside-2-phosphate, and it was examined whether the CK reaction proceeded in the presence of both.
- a reaction solution having the following composition was prepared. 50 mmol / L buffer PIPES (pH 7.0) 10 mmol / L MgCl 2 10 mmol / L glucose 1 mmol / L ADP 1mmol / L IDP 1mmol / L NADP 8mmol / L N-acetylcysteine (NAC) 1u / mL G6PDH 1u / mL ADP-HKII
- a reaction solution having the following composition was prepared. 50 mmol / L buffer PIPES (pH 7.0) 10 mmol / L MgCl 2 10 mmol / L glucose 2 mmol / L ATP 1mmol / L IDP 1mmol / L NADP 8mmol / L N-acetylcysteine (NAC) 1u / mL G6PDH 1u / mL ADP-HKII
- CK manufactured by Asahi Kasei Pharma Corporation, HC-CKII, catalog No. T-744 was added to 100 mL / mL of the reaction solution. Preheating at 37 ° C. and adding 0.05 mL of 0 mmol / L, 0.2 mmol / L, 0.4 mmol / L, 1, 2 mmol / L creatine solution increases absorbance at 340 nm in proportion to time did. Therefore, the amount of change in absorbance from the 3rd minute to the 5th minute at 340 nm was recorded. The results are shown in FIG. As is clear from FIG. 1, the amount of change in absorbance was proportional to the creatine concentration in the sample.
- a reaction solution having the following composition was prepared. 50 mmol / L buffer PIPES (pH 7.0) 10 mmol / L MgCl 2 10mmol / L glucose 1mmol / L NADP 1 mmol / L nucleoside-3-phosphate 4 mmol / L nucleoside-2-phosphate 8 mmol / L N-acetylcysteine (NAC) 1u / mL G6PDH 1u / mL ADP-HKII 100u / mL CK (derived from rabbit muscle)
- dATP / IDP, dATP / GDP, and dATP / dGDP are selected as combinations of nucleoside-3-phosphate (1 mmol / L) and nucleoside-2-phosphate (4 mmol / L).
- nucleoside-3-phosphate 1 mmol / L
- nucleoside-2-phosphate 4 mmol / L
- a reaction solution having the following composition was prepared. 50 mmol / L buffer PIPES (pH 7.0) 10 mmol / L MgCl 2 10 mmol / L glucose 1 mmol / L NAD 1mmol / L ATP 4mmol / L IDP 8mmol / L N-acetylcysteine (NAC) 1u / mL G6PDH 1u / mL ADP-HKII 250u / mL CK (derived from rabbit muscle)
- Reaction liquid 1 having the following composition was prepared. 50 mmol / L PIPES-NaOH buffer (pH 7.0) 12 mmol / L MgCl 2 10 mmol / L glucose 5.3 mmol / L IDP 20u / mL creatinine amide hydrolase (Toyobo)
- reaction liquid 2 having the following composition was prepared. 200 mmol / L buffer PIPES (pH 7.0) 2 mmol / L MgCl 2 4mmol / L Thio NAD 4mmol / L ATP 10 mmol / L N-acetylcysteine (NAC) 4u / mL G6PDH 4u / mL ADP-HKII 1000u / mL CK (from rabbit muscle)
- Example 6 Determination of 3-phosphoglyceric acid
- a reaction solution having the following composition was prepared. 50 mmol / L PIPES-NaOH buffer (pH 7.5) 10 mmol / L MgCl 2 10 mmol / L glucose 1 mmol / L ADP-HKPII 1u / mL G6PDH 2mmol / L NAD 1mmol / L ATP 4mmol / L IDP 20 u / mL 3-phosphoglycerate kinase (PGK) (manufactured by Sigma: derived from yeast)
- PGK 3-phosphoglycerate kinase
- Example 7 Determination of fructose-6-phosphate with phosphofructo-1-kinase (PFK)
- PFK phosphofructo-1-kinase
- fructose-6-phosphate solution 0 mL / L, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L of fructose-6-phosphate solution was added to 1 mL of the above reaction solution. .02 mL was added and prewarmed at 37 ° C. For each, phosphofructo-1-kinase (Asahi Kasei Pharma Co., Ltd., T-142: Geobacillus stearothermophilus derived) was added at 30 u / mL, and the reaction was started at 37 ° C.
- Example 8 Determination of glycerol by GK
- a reaction solution having the following composition was prepared.
- glycerol kinase a microorganism-derived enzyme was used as described below. 50 mmol / L PIPES-NaOH buffer (pH 7.5) 5 mmol / L MgCl 2 10 mmol / L glucose 1 mmol / L ADP-HKPII 1u / mL G6PDH 1mmol / L NAD 0.5mmol / L ATP 4mmol / L IDP 20u / mL Glycerol kinase (Asahi Kasei Pharma Co., Ltd., T-64: derived from Flavobacterium meningosepticum)
- Example 9 Effect of adding PK IDP
- a reaction solution having the following composition was prepared using pyruvate kinase derived from rabbit muscle. 50 mmol / L PIPES-NaOH buffer (pH 7.5) 5 mmol / L magnesium chloride (MgCl 2 ) 10 mmol / L glucose 1 mmol / L ADP-HKPII 1u / mL G6PDH 1mmol / L NAD 3mmol / L ATP 10u / mL pyruvate kinase (PK, Sigma, derived from rabbit muscle)
- PGK 3-phosphoglycerate kinase
- GK glycerokinase
- Example 11 Determination of glucose 6-phosphate with hexokinase
- a reaction solution having the following composition was prepared. 50 mmol / L PIPES buffer (pH 7.0) 5 mmol / L MgCl 2 0.5mmol / L ADP 10 mmol / L Glycerol 0.3% 4-Aminoantipyrine 20u / mL GPOSP (Asahi Kasei Pharma Corporation, Catalog No. T-60) 0.2% TOOS (Dojin Chemical) 4.5u / mL peroxidase (Sigma) 1u / mL glycerokinase (from E. coli: Sigma) 50u / mL HKIII (Asahi Kasei Pharma Corporation; catalog No. T-141)
- the principle of measurement is to specifically quantify ATP produced by the HKIII reaction using glycerokinase derived from E. coli in the presence of ADP and GTP.
- a glucose-6-phosphate solution was added to 1 mL of the reaction solution so that the concentration in the reaction solution was 1 ⁇ mol / L, 2 ⁇ mol / L, 3 ⁇ mol / L, 4 ⁇ mol / L, and 5 ⁇ mol / L, and pre-warmed.
- GTP aqueous solution was added so that the density
- Example 12 Reverse reaction of ADP-dependent glucokinase
- a reaction solution having the following composition was prepared. 50 mmol / L PIPES buffer (pH 7.0) 5 mmol / L MgCl 2 5mmol / L creatine phosphate 0.5mmol / L AMP 1mmol / L CDP 0.5mmol / L deamido-NAD 20 mmol / L NH 4 Cl 1 mmol / L sodium cholate 0.5 u / mL NAD synthase (NADSII: Asahi Kasei Pharma Corporation, T-67) 2u / mL 12 ⁇ -hydroxysteroid dehydrogenase (12 ⁇ -HSDII: Asahi Kasei Pharma Corporation, T-190) 10u / mL CK (HC-CKII: Asahi Kasei Pharma Corporation, T-74)
- ADP-HKPII (Asahi Kasei Pharma Corporation: T-92) was used as the ADP-dependent glucokinase, and CDP and AMP were selected as the two nucleotide coenzymes.
- ADP was first converted to ATP with CK, then converted to NAD with NAD synthase, and finally the production rate of reduced NAD by 12 ⁇ -HSDII was 340 nm. Detected.
- Example 13 Determination of phosphoenolpyruvate by PK
- a reaction solution having the following composition was prepared. 50 mmol / L Tris-HCl buffer (pH 9.0) 5mmol / L magnesium chloride 10 mmol / L glucose 50 mmol / L potassium chloride 1 u / mL ADP-HK (manufactured by Asahi Kasei Pharma Corporation: T-92) 1u / mL G6PDH (manufactured by Toyobo) 1mmol / L NAD 2mmol / L ATP 50u / mL pyruvate kinase (PK, Sigma, rabbit muscle origin)
- reaction liquid 1 having the following composition was prepared. 50 mmol / L TES buffer (pH 7.5) 12 mmol / L Magnesium chloride 12 mmol / L Glucose 1.5 u / mL ADP-HK (Asahi Kasei Pharma Co., Ltd .: T-92) 1.5u / mL G6PDH (Toyobo) 1.5mmol / L Thio NAD (Oriental Yeast Industry) 0.7mmol / L ATP
- reaction solution 2 having the following composition was prepared. 50 mmol / L TES buffer (pH 7.5) 8mmol / L IDP 0.5 mmol / L N-acetylcysteine 0.02% BSA 16u / mL creatinine amide hydrolase (Toyobo) 200u / mL CK-B (human-derived B-type isozyme)
- a conversion reaction from creatinine to creatine by creatinine amide hydrolase and a CK cycling reaction were simultaneously performed.
- 0.05 mL of 0.04 mmol / L, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L creatinine aqueous solution was added to 0.75 mL of reaction solution 1, and pre-warmed at 37 ° C. for 3 minutes. .
- 0.25 mL of the reaction solution 2 was added to start the reaction.
- the change in absorbance at 400 nm from the 3rd minute to the 4th minute after the addition of the reaction solution 2 was measured. As a result, as shown in FIG. 12, the amount of change in absorbance increased quantitatively according to the creatinine concentration.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Emergency Medicine (AREA)
- Sustainable Development (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
[1]キナーゼの正反応基質、そのリン酸化物、及びそれらへ誘導される誘導前物質の少なくとも一つの測定方法であって、
(1)キナーゼであって、ヌクレオチド補酵素の存在下、キナーゼの正反応基質からそのリン酸化物を生成する正反応、及びその逆反応を触媒し、正反応と逆反応において、少なくともそれぞれ別異のヌクレオシド部分を有するヌクレオチド補酵素を利用するキナーゼと、キナーゼの第一のヌクレオチド補酵素と、第一のヌクレオチド補酵素とはヌクレオシド部分が異なる第二のヌクレオチド補酵素と、を
試料、又は測定対象が誘導前物質である場合には誘導前物質をキナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理を行った試料と接触せしめ、下記式(1)のサイクリング反応を反応せしめる工程と、
(2)第一のヌクレオチド補酵素、第一のヌクレオチド補酵素の変化物、第二のヌクレオチド補酵素、及び第二のヌクレオチド補酵素の変化物の少なくともいずれかの変化量に対応するシグナルの変化量を検出する工程と、
(3)検出されたシグナルの変化量に基づき、試料が含有するキナーゼの正反応基質、そのリン酸化物、及びそれらへ誘導される誘導前物質の少なくとも一つの量を算出する工程と、
を含む測定方法。
測定対象がキナーゼの正反応基質及び/又はそのリン酸化物である、上記[1]に記載の測定方法。
測定対象がキナーゼ正反応基質及び/又はそのリン酸化物であって、測定対象とキナーゼの組み合わせが以下のいずれかである、上記[1]から[1-1-1]のいずれかに記載の測定方法。なお以下においては、測定対象:キナーゼの順に記載されている。また、そのリン酸化物とは、記載されたキナーゼ正反応基質に対応するリン酸化物である。
クレアチン/そのリン酸化物:クレアチンキナーゼ(EC 2.7.3.2)
3-ホスホグリセリン酸/そのリン酸化物:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3)
ピルビン酸/そのリン酸化物:ピルビン酸キナーゼ(EC 2.7.1.40)
フルクトース-6-リン酸/そのリン酸化物:ホスホフルクト-1-キナーゼ(EC 2.7.1.11)
グリセロール/そのリン酸化物:グリセロールナーゼ(EC 2.7.1.30)
ヘキソース/そのリン酸化物:ヘキソキナーゼ(EC 2.7.1.1)
グルコース/そのリン酸化物:ADP依存性グルコキナーゼ(EC 1.7.1.147)
測定対象が誘導前物質であり、試料が、誘導前物質を、キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理を行った試料であり、検出されたシグナルの変化量に基づき、誘導前物質の量が算出される、上記[1]に記載の測定方法。
測定対象が誘導前物質であり、測定対象、キナーゼ、並びにキナーゼの正反応基質及び/又はそのリン酸化物の組み合わせが、表2に記載のいずれかである、上記[1]又は[1-2]に記載の測定方法。
測定対象が、誘導前物質であって、測定対象、キナーゼ、及びキナーゼ正反応基質/そのリン酸化物の組み合わせが以下の組み合わせのいずれかである、上記[1]、[1-2]及び[1-2-1]のいずれかに記載の測定方法。なお以下においては、測定対象:キナーゼ:キナーゼ正反応基質の順に記載されている。
クレアチニン:クレアチンキナーゼ(EC 2.7.3.2):クレアチン/そのリン酸化物
グリセロアルデヒド-3-リン酸、ヒドロキシアセトンリン酸、グリセロール-3-リン酸、リゾホスファチジン酸、リゾホスファチジルコリン、フルクトース-1,6-ビスリン酸、2-ホスホグリセリン酸、ホスホエノールピルビン酸、又は2,3-ビスホスホグリセリン酸:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3):3-ホスホグリセリン酸/そのリン酸化物
グルコース-6-リン酸:ホスホフルクト-1-キナーゼ(EC 2.7.1.11):ホスホフルクトース-6-リン酸/そのリン酸化物
ドロキシアセトンリン酸、トリグリセリド、リゾホスファチジルコリン、リゾホスファチジン酸、グリセロール-3-リン酸、リゾホスファチジルグリセロール、又はホスファチジルグリセロール: グリセロキナーゼ(EC 2.7.1.30): グリセロール/そのリン酸化物
グルコース-1-リン酸: ADP依存性グルコキナーゼ(EC 2.7.1.147):グルコース/そのリン酸化物
測定対象が誘導前物質であるクレアチニン、キナーゼがクレアチンキナーゼ、キナーゼの正反応基質がクレアチンである、上記[1]、[1-2]及び[1-2-2]のいずれかに記載の測定方法。
誘導前物質を、キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理において、クレアチニンに、水の存在下、クレアチニンアミドヒドロラーゼ(EC 3.5.2.10)を接触させることを含む、上記[1]、及び[1-2]ないし[1-2-3]のいずれかに記載の測定方法。
上記工程(2)が、第一のヌクレオチド補酵素の正反応変化物、又は第二のヌクレオチド補酵素の逆反応変化物の増加量に対応するシグナルの変化量を検出する工程である、上記[1]から[1-2-4]のいずれかに記載の測定方法。
第一のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン、及びデオキシイノシンのいずれかであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[1]~[2]のいずれかに記載の測定方法。
第一のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、イノシン、グアノシン、又はデオキシアデノシンであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[1]~[3]に記載の測定方法。
第二のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン及びデオキシイノシンのいずれかであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[1]から[4]のいずれかに記載の測定方法。
第二のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、イノシン、グアノシン、又はデオキシグアノシンであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[1]から[5]のいずれかに記載の測定方法。
第一のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、イノシン、グアノシン、又はデオキシアデノシンであって、第二のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、イノシン、グアノシン、又はデオキシグアノシンであり、かつ、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[1]から[6]のいずれかに記載の測定方法。
第一のヌクレオチド補酵素のヌクレオシド部分と第二のヌクレオチド補酵素のヌクレオシド部分の組み合わせが、アデノシンとイノシン、グアノシンとアデノシン、デオキシアデノシンとグアノシン、デオキシアデノシンとデオキシグアノシン、デオキシアデノシンとイノシン、又はイノシンとアデノシンである、上記[1]から[7]のいずれかに記載の測定方法。
第一のヌクレオチド補酵素のヌクレオシド部分がアデノシンであって、第二のヌクレオチド補酵素のヌクレオシド部分が、イノシンである、上記[1]から[7-1]のいずれかに記載の測定方法。
第一のヌクレオチド補酵素がアデノシン3リン酸(ATP)であり、第二のヌクレオチド補酵素がイノシン2リン酸(IDP)である、上記[1]ないし[7-3]のいずれかに記載の測定方法。
シグナルの変化量を検出する工程において、正反応による第一のヌクレオチド補酵素の変化物を利用できるが、第二のヌクレオチド補酵素を利用できない検出用酵素、又は第一のヌクレオチド補酵素を利用できないが、逆反応による第二のヌクレオチド補酵素の変化物を利用できる検出用酵素を用いて、正反応による第一のヌクレオチド補酵素の変化物又は逆反応による第二のヌクレオチド補酵素の変化物の増加量に対応するシグナルの変化量を検出する、上記[1]ないし[7-3]のいずれかに記載の測定方法。
シグナルの変化量を検出する工程において、アデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 2.7.1.147)を用いて、グルコースの存在下、アデノシン2リン酸(ADP)の増加量に対応して変化するシグナルの変化量を検出する、上記[1]から[8]のいずれかに記載の測定方法。
シグナルの変化量を検出する工程において、アデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 2.7.1.147)を用いて、チオニコチンアミドアデニンジヌクレオチドリン酸(チオNADP)、チオニコチンアミドアデニンジヌクレオチド(チオNAD)、ニコチンアミドアデニンジヌクレオチドリン酸(NADP)、及びニコチンアミドアデニンジヌクレオチド(NAD)のいずれかの補酵素と、グルコースと、グルコース6リン酸デヒドロゲナーゼと、の存在下、アデノシン2リン酸(ADP)の増加量に対応して変化するシグナルの変化量を検出する、上記[1]ないし[9]のいずれかに記載の測定方法。
測定対象が誘導前物質であるクレアチニンの測定方法であって、以下の工程を含む、上記[1]及び[1-2]から[10]のいずれかに記載の測定方法。
(1)クレアチニンに、水の存在下、クレアチニン アミドヒドロラーゼ(EC3.5.2.10)を接触させ、クレアチンを定量的に誘導せしめる処理を行った試料、と、少なくとも以下の成分(a)~(c)とを接触せしめ、下記式(2)のサイクリング反応を反応せしめる工程と、
(a)クレアチンキナーゼ(EC 2.7.3.2)
(b)ATP
(c)IDP
(2)アデノシン2リン酸依存性グルコキナーゼ(EC 2.7.1.147)を用いて、チオNADP、チオNAD、NADP、又はNADのいずれかの補酵素と、グルコースと、グルコース6リン酸デヒドロゲナーゼと、の存在下、アデノシン2リン酸の増加量に対応するシグナルの変化量を検出する工程と、
(3)上記(2)の工程の検出結果から、試料中のクレアチニンの量を算出する工程。
キナーゼの正反応基質、そのリン酸化物、及びそれらへ誘導される誘導前物質の少なくとも一つを測定するための測定用組成物であって、測定対象が誘導前物質である場合には、誘導前物質をキナーゼ正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理をした後に用いられ、且つ
(a)キナーゼであって、ヌクレオチド補酵素の存在下、当該キナーゼの正反応基質からそのリン酸化物を生成する正反応、及びその逆反応を触媒し、正反応と逆反応において、少なくともそれぞれ別異のヌクレオシド部分を有するヌクレオチド補酵素を利用するキナーゼと、
(b)正反応における第一のヌクレオチド補酵素と、
(c)第一のヌクレオチド補酵素とはヌクレオシド部分が異なる、逆反応における第二のヌクレオチド補酵素と、
を備える、組成物。
測定対象がキナーゼ正反応基質及び/又はそのリン酸化物である、上記[12]に記載の組成物。
測定対象がキナーゼ正反応基質及び/又はそのリン酸化物であって、測定対象とキナーゼの組み合わせが以下の組み合わせのいずれかである、上記[12]から[12-1-1]のいずれかに記載の組成物。なお以下においては、測定対象:キナーゼの順に記載されている。また、そのリン酸化物とは、記載されたキナーゼ正反応基質に対応するリン酸化物である。
クレアチン/そのリン酸化物:クレアチンキナーゼ(EC 2.7.3.2)
3-ホスホグリセリン酸/そのリン酸化物:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3)
ピルビン酸/そのリン酸化物:ピルビン酸キナーゼ(EC 2.7.1.40)
フルクトース-6-リン酸/そのリン酸化物:ホスホフルクト-1-キナーゼ(EC 2.7.1.11)
グリセロール:グリセロールナーゼ(EC 2.7.1.30)
ヘキソース/そのリン酸化物:ヘキソキナーゼ(EC 2.7.1.1)
グルコース/そのリン酸化物:ADP依存性グルコキナーゼ(EC 2.7.1.147)
測定対象が誘導前物質であり、当該誘導前物質を、キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理をした後に用いられる、上記[12]に記載の組成物。
測定対象が誘導前物質であり、測定対象、キナーゼ、並びにキナーゼの正反応基質及び/又はそのリン酸化物の組み合わせが、表4に記載のいずれかである、上記[12]又は[12-2]に記載の組成物。
測定対象が誘導前物質であって、測定対象、キナーゼ、及びキナーゼ正反応基質/そのリン酸化物の組み合わせが、以下の組み合わせのいずれかである、上記[12]、[12-2]及び[12-2-1]のいずれかに記載の組成物。なお以下においては、測定対象:キナーゼ:キナーゼ正反応基質/そのリン酸化物の順に記載されている。
クレアチニン:クレアチンキナーゼ(EC 2.7.3.2):クレアチン/そのリン酸化物
グリセロアルデヒド-3-リン酸、ヒドロキシアセトンリン酸、グリセロール-3-リン酸、リゾホスファチジン酸、リゾホスファチジルコリン、フルクトース-1,6-ビスリン酸、2-ホスホグリセリン酸、ホスホエノールピルビン酸、又は2,3-ビスホスホグリセリン酸:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3):3-ホスホグリセリン酸/そのリン酸化物
グルコース-6-リン酸:ホスホフルクト-1-キナーゼ(EC 2.7.1.11):ホスホフルクトース-6-リン酸/そのリン酸化物)、(ジヒドロキシアセトンリン酸、トリグリセリド、リゾホスファチジルコリン、リゾホスファチジン酸、グリセロール-3-リン酸、リゾホスファチジルグリセロール、又はホスファチジルグリセロール:グリセロキナーゼ(EC 2.7.1.30):グリセロール/そのリン酸化物
グルコース-1-リン酸:ADP依存性グルコキナーゼ(EC 2.7.1.147):グルコース/そのリン酸化物
測定対象が誘導前物質であるクレアチニン、キナーゼがクレアチンキナーゼ、前記キナーゼの正反応基質がクレアチンである、上記[12]及び[12-2]から[12-2-2]のいずれかに記載の組成物。
誘導前物質を、キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理において、クレアチニンに、水の存在下、クレアチニンアミドヒドロラーゼ(EC3.5.2.10)を接触させ、クレアチンを誘導せしめる、上記[12]、[12-2]から[12-2-3]のいずれかに記載の組成物。
第一のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン及びデオキシイノシンのいずれかであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[12]から[12-2-4]のいずれかに記載の組成物。
第一のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、イノシン、グアノシン、又はデオキシアデノシンであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[12]から[13]のいずれかに記載の組成物。
第二のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン及びデオキシイノシンのいずれかであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[12]から[14]のいずれかに記載の組成物。
第二のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、イノシン、グアノシン、又はデオキシグアノシンであり、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[12]から[15]のいずれかに記載の組成物。
第一のヌクレオチド補酵素のヌクレオシド部分がアデノシン、イノシン、グアノシン、又はデオキシアデノシンであって、第二のヌクレオチド補酵素のヌクレオシド部分がイノシン、グアノシン、アデノシン、又はデオキシグアノシンであり、かつ、第一のヌクレオチド補酵素と第二のヌクレオチド補酵素のヌクレオシド部分が別異である、上記[12]から[16]のいずれかに記載の組成物。
第一のヌクレオチド補酵素のヌクレオシド部分と第二のヌクレオチド補酵素のヌクレオシド部分の組み合わせが、アデノシンとイノシン、グアノシンとアデノシン、デオキシアデノシンとグアノシン、デオキシアデノシンとデオキシグアノシン、デオキシアデノシンとイノシン、又はグアノシンとアデノシンである、上記[12]から[17]のいずれかに記載の組成物。
第一のヌクレオチド補酵素のヌクレオシド部分がアデノシンであり、第二のヌクレオチド補酵素のヌクレオシド部分がイノシンである、上記[12]から[17-1]のいずれかに記載の組成物。
第一のヌクレオチド補酵素がアデノシン3リン酸(ATP)であり、第二のヌクレオチド補酵素がイノシン2リン酸(IDP)である、上記[12]から[17-2]のいずれかに記載の組成物。
正反応による第一のヌクレオチド補酵素の変化物を利用できるが、第二のヌクレオチド補酵素を利用できない検出用酵素、又は第一のヌクレオチド補酵素を利用できないが、逆反応による第二のヌクレオチド補酵素の変化物は利用できる検出用酵素を更に含む、上記[12]から[17-3]のいずれかに記載の組成物。
検出用酵素がアデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 1.7.1.147)であり、グルコースを更に含む、上記[18]に記載の組成物。
チオニコチンアミドアデニンジヌクレオチドリン酸(チオNADP)、チオニコチンアミドアデニンジヌクレオチド(チオNAD)、ニコチンアミドアデニンジヌクレオチドリン酸(NADP)又はニコチンアミドアデニンジヌクレオチド(NAD)と、
グルコース6リン酸デヒドロゲナーゼと、
を更に含む、上記[18]に記載の組成物。
測定対象が誘導前物質であるクレアチニンであり、
当該クレアチニンに、水の存在下、クレアチニン アミドヒドロラーゼ(EC3.5.2.10)を接触させ、クレアチニンをクレアチンへと定量的に誘導せしめる処理をした後に用いられ、
(a)クレアチンキナーゼと、
(b)アデノシン3リン酸(ATP)と、
(c)イノシン2リン酸(IDP)と、
(d)アデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 2.7.1.147)と、
(e)グルコースと、
(f)チオニコチンアミドアデニンジヌクレオチドリン酸(チオNADP)、チオニコチンアミドアデニンジヌクレオチド(チオNAD)、ニコチンアミドアデニンジヌクレオチドリン酸(NADP)又はニコチンアミドアデニンジヌクレオチド(NAD)と、
(g)グルコース6リン酸デヒドロゲナーゼと、
を備える、上記[12-2]ないし[20]のいずれかに記載の組成物。
上記[12]から[21]のいずれかに記載の組成物を含む、試薬キット。
(1)キナーゼであって、ヌクレオチド補酵素の存在下、キナーゼの正反応基質からそのリン酸化物を生成する正反応、及びその逆反応を触媒し、正反応と逆反応において、少なくともそれぞれ別異のヌクレオシド部分を有するヌクレオチド補酵素を利用するキナーゼと、キナーゼの第一のヌクレオチド補酵素と、第一のヌクレオチド補酵素とはヌクレオシド部分が異なる第二のヌクレオチド補酵素と、を、試料、又は測定対象が誘導前物質である場合には誘導前物質をキナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理を行った試料と接触せしめ、下記式(3)の酵素サイクリング反応を反応せしめる工程と、
(2)第一のヌクレオチド補酵素、第一のヌクレオチド補酵素の変化物、第二のヌクレオチド補酵素、及び第二のヌクレオチド補酵素の変化物の少なくともいずれかの変化量に対応するシグナルの変化量を検出する工程と、
(3)検出されたシグナルの変化量に基づき、試料が含有するキナーゼの正反応基質、そのリン酸化物、及びそれらへ誘導される誘導前物質の少なくとも一つの量を算出する工程と、
を含む測定方法である。
クレアチン及び/又はそのリン酸化物:クレアチンキナーゼ
3-ホスホグリセリン酸及び/又はそのリン酸化物:3-ホスホグリセリン酸キナーゼ
ピルビン酸及び/又はそのリン酸化物:ピルビン酸キナーゼ
フルクトース-6-リン酸及び/又はそのリン酸化物:ホスホフルクト-1-キナーゼ
グリセロール及び/又はそのリン酸化物:グリセロールキナーゼ
ヘキソース及び/又はそのリン酸化物:ヘキソキナーゼ
グルコース及び/又はそのリン酸化物:アデノシン2リン酸(ADP)依存性グルコキナーゼ
クレアチン及び/又はそのリン酸化物:クレアチンキナーゼ(EC 2.7.3.2)
3-ホスホグリセリン酸及び/又はそのリン酸化物:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3)
ピルビン酸及び/又はそのリン酸化物:ピルビン酸キナーゼ(EC 2.7.1.40)
フルクトース-6-リン酸及び/又はそのリン酸化物:ホスホフルクト-1-キナーゼ(EC 2.7.1.11)
グリセロール及び/又はそのリン酸化物:グリセロールナーゼ(EC 2.7.1.30)
ヘキソース及び/又はそのリン酸化物:ヘキソキナーゼ(EC 2.7.1.1)
グルコース及び/又はそのリン酸化物:ADP依存性グルコキナーゼ(EC 2.7.1.147)
クレアチニン:クレアチンキナーゼ:クレアチン及び/又はそのリン酸化物
グリセロアルデヒド-3-リン酸、ヒドロキシアセトンリン酸、グリセロール-3-リン酸、リゾホスファチジン酸、リゾホスファチジルコリン、フルクトース-1,6-ビスリン酸、2-ホスホグリセリン酸、ホスホエノールピルビン酸、又は2,3-ビスホスホグリセリン酸:3-ホスホグリセリン酸キナーゼ:3-ホスホグリセリン酸及び/又はそのリン酸化物
グルコース-6-リン酸:ホスホフルクト-1-キナーゼ:ホスホフルクトース-6-リン酸及び/又はそのリン酸化物
ジヒドロキシアセトンリン酸、トリグリセリド、リゾホスファチジルコリン、リゾホスファチジン酸、グリセロール-3-リン酸、リゾホスファチジルグリセロール、又はホスファチジルグリセロール:グリセロキナーゼ:グリセロール及び/又はそのリン酸化物
グルコース-1-リン酸:ADP依存性グルコキナーゼ:グルコース及び/又はそのリン酸化物
クレアチニン:クレアチンキナーゼ(EC 2.7.3.2):クレアチン及び/又はそのリン酸化物
グリセロアルデヒド-3-リン酸、ヒドロキシアセトンリン酸、グリセロール-3-リン酸、リゾホスファチジン酸、リゾホスファチジルコリン、フルクトース-1,6-ビスリン酸、2-ホスホグリセリン酸、ホスホエノールピルビン酸、又は2,3-ビスホスホグリセリン酸:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3):3-ホスホグリセリン酸及び/又はそのリン酸化物
グルコース-6-リン酸:ホスホフルクト-1-キナーゼ(EC 2.7.1.11):ホスホフルクトース-6-リン酸及び/又はそのリン酸化物
ドロキシアセトンリン酸、トリグリセリド、リゾホスファチジルコリン、リゾホスファチジン酸、グリセロール-3-リン酸、リゾホスファチジルグリセロール、又はホスファチジルグリセロール:グリセロキナーゼ(EC 2.7.1.30):グリセロール及び/又はそのリン酸化物
グルコース-1-リン酸:ADP依存性グルコキナーゼ(EC 2.7.1.147):グルコース及び/又はそのリン酸化物
クレアチニン:クレアチンキナーゼ:クレアチン及び/又はそのリン酸化物
(a)キナーゼであって、ヌクレオチド補酵素の存在下、当該キナーゼの正反応基質からそのリン酸化物を生成する正反応、及びその逆反応を触媒し、正反応と逆反応において、少なくともそれぞれ別異のヌクレオシド部分を持つヌクレオチド補酵素を利用するキナーゼと、
(b)正反応における第一のヌクレオチド補酵素と、
(c)第一のヌクレオチド補酵素とはヌクレオシド部分が異なる、逆反応における第二のヌクレオチド補酵素と、
を備える組成物であって、
測定対象が誘導前物質である場合には、誘導前物質をキナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理をした後に用いる組成物である。
クレアチン及び/又はそのリン酸化物:クレアチンキナーゼ
3-ホスホグリセリン酸及び/又はそのリン酸化物:3-ホスホグリセリン酸キナーゼ
ピルビン酸及び/又はそのリン酸化物:ピルビン酸キナーゼ
フルクトース-6-リン酸及び/又はそのリン酸化物:ホスホフルクト-1-キナーゼ
グリセロール及び/又はそのリン酸化物:グリセロールキナーゼ
ヘキソース及び/又はそのリン酸化物:ヘキソキナーゼ
グルコース及び/又はそのリン酸化物:ADP依存性グルコキナーゼ
クレアチン及び/又はそのリン酸化物:クレアチンキナーゼ(EC 2.7.3.2)
3-ホスホグリセリン酸及び/又はそのリン酸化物:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3)
ピルビン酸及び/又はそのリン酸化物:ピルビン酸キナーゼ(EC 2.7.1.40)
フルクトース-6-リン酸及び/又はそのリン酸化物:ホスホフルクト-1-キナーゼ(EC 2.7.1.11)
グリセロール及び/又はそのリン酸化物:グリセロールナーゼ(EC 2.7.1.30)
ヘキソース及び/又はそのリン酸化物:ヘキソキナーゼ(EC 2.7.1.1)
グルコース及び/又はそのリン酸化物:ADP依存性グルコキナーゼ(EC 2.7.1.147)
クレアチニン:クレアチンキナーゼ:クレアチン及び/又はそのリン酸化物
グリセロアルデヒド-3-リン酸、ヒドロキシアセトンリン酸、グリセロール-3-リン酸、リゾホスファチジン酸、リゾホスファチジルコリン、フルクトース-1,6-ビスリン酸、2-ホスホグリセリン酸、ホスホエノールピルビン酸、又は2,3-ビスホスホグリセリン酸:3-ホスホグリセリン酸キナーゼ:3-ホスホグリセリン酸及び/又はそのリン酸化物
グルコース-6-リン酸:ホスホフルクト-1-キナーゼ:ホスホフルクトース-6-リン酸及び/又はそのリン酸化物
ジヒドロキシアセトンリン酸、トリグリセリド、リゾホスファチジルコリン、リゾホスファチジン酸、グリセロール-3-リン酸、リゾホスファチジルグリセロール、又はホスファチジルグリセロール:グリセロキナーゼ:グリセロール及び/又はそのリン酸化物
グルコース-1-リン酸:ADP依存性グルコキナーゼ:グルコース及び/又はそのリン酸化物
クレアチニン:クレアチンキナーゼ(EC 2.7.3.2):クレアチン及び/又はそのリン酸化物
グリセロアルデヒド-3-リン酸、ヒドロキシアセトンリン酸、グリセロール-3-リン酸、リゾホスファチジン酸、リゾホスファチジルコリン、フルクトース-1,6-ビスリン酸、2-ホスホグリセリン酸、ホスホエノールピルビン酸、又は2,3-ビスホスホグリセリン酸:3-ホスホグリセリン酸キナーゼ(EC 2.7.2.3):3-ホスホグリセリン酸及び/又はそのリン酸化物
グルコース-6-リン酸:ホスホフルクト-1-キナーゼ(EC 2.7.1.11):ホスホフルクトース-6-リン酸/そのリン酸化物)、(ドロキシアセトンリン酸、トリグリセリド、リゾホスファチジルコリン、リゾホスファチジン酸、グリセロール-3-リン酸、リゾホスファチジルグリセロール、又はホスファチジルグリセロール:グリセロキナーゼ(EC 2.7.1.30):グリセロール及び/又はそのリン酸化物
グルコース-1-リン酸:ADP依存性グルコキナーゼ(EC 2.7.1.147):グルコース及び/又はそのリン酸化物
クレアチニン:クレアチンキナーゼ:クレアチン及び/又はそのリン酸化物
Pyrococcus furiosus由来のADP依存性ヘキソキナーゼ(ADP-HKPII(T-92) 旭化成ファーマ株式会社)が、ヌクレオシド-2-リン酸としてADPの他にIDP、GDPをリン酸供与体とすることができないことを確認した。すなわち、ADP-HKPII反応により生成するグルコースを、NADPの存在下、グルコース-6-リン酸デヒドロゲナーゼ(G6PDH:東洋紡)を共存させ、37℃にて還元型NADPに基づく340nmの吸光度変化として測定した。
50mmol/L Tris-HCl pH8.0
10mmol/L グルコース
2mmol/L 塩化マグネシウム(MgCl2)
1mmol/L NADP
1u/mL G6PDH
1mmol/L ヌクレオシド-2-リン酸(ADP、IDP、又はGDP)
上記のように、ADP-HKPIIが実質的にIDPを水素供与体としないことが確認されたため、第一のヌクレオチド補酵素としてのヌクレオシド-3-リン酸にATPを、第二のヌクレオチド補酵素としてのヌクレオシド-2-リン酸にIDPを選択し、両者の存在下にCK反応が進行するかどうかを調べた。
10mmol/L MgCl2
10mmol/L グルコース
1mmol/L ADP
1mmol/L IDP
1mmol/L NADP
8mmol/L N-アセチルシステイン(NAC)
1u/mL G6PDH
1u/mL ADP-HKII
クレアチンリン酸+IDP→クレアチン+ITP
クレアチン+ATP→クレアチン+ADP
下記の組成の反応液を調製した。 50mmol/L 緩衝剤PIPES (pH7.0)
10mmol/L MgCl2
10mmol/L グルコース
2mmol/L ATP
1mmol/L IDP
1mmol/L NADP
8mmol/L N-アセチルシステイン(NAC)
1u/mL G6PDH
1u/mL ADP-HKII
下記の組成の反応液を調製した。
50mmol/L 緩衝剤PIPES (pH7.0)
10mmol/L MgCl2
10mmol/L グルコース
1mmol/L NADP
1mmol/L ヌクレオシド-3-リン酸
4mmol/L ヌクレオシド-2-リン酸
8mmol/L N-アセチルシステイン(NAC)
1u/mL G6PDH
1u/mL ADP-HKII
100u/mL CK (ウサギ筋肉由来)
下記の組成の反応液を調製した。
50mmol/L 緩衝剤PIPES (pH7.0)
10mmol/L MgCl2
10mmol/L グルコース
1mmol/L NAD
1mmol/L ATP
4mmol/L IDP
8mmol/L N-アセチルシステイン(NAC)
1u/mL G6PDH
1u/mL ADP-HKII
250u/mL CK (ウサギ筋肉由来)
下記の組成の反応液1を調製した。
50mmol/L PIPES-NaOH 緩衝液(pH7.0)
12mmol/L MgCl2
10mmol/L グルコース
5.3mmol/L IDP
20u/mL クレアチニンアミドヒドロラーゼ(東洋紡製)
200mmol/L 緩衝剤PIPES (pH7.0)
2mmol/L MgCl2
4mmol/L チオNAD
4mmol/L ATP
10mmol/L N-アセチルシステイン(NAC)
4u/mL G6PDH
4u/mL ADP-HKII
1000u/mL CK (ウサギ筋肉由来)
下記の組成の反応液を調製した。
50mmol/L PIPES-NaOH緩衝液(pH7.5)
10mmol/L MgCl2
10mmol/L グルコース
1mmol/L ADP-HKPII
1u/mL G6PDH
2mmol/L NAD
1mmol/L ATP
4mmol/L IDP
20u/mL 3-ホスホグリセリン酸キナーゼ(PGK)(シグマ社製:酵母由来)
下記の組成の反応液を調製した。
50mmol/L PIPES-NaOH緩衝液(pH7.5)
5mmol/L MgCl2
10mmol/L グルコース
1mmol/L ADP-HKPII
1u/mL G6PDH
1mmol/L NAD
0.5mmol/L ATP
5mmol/L IDP
下記の組成の反応液を調製した。グリセロールキナーゼとしては、下記のように微生物由来の酵素を用いた。
50mmol/L PIPES-NaOH緩衝液(pH7.5)
5mmol/L MgCl2
10mmol/L グルコース
1mmol/L ADP-HKPII
1u/mL G6PDH
1mmol/L NAD
0.5mmol/L ATP
4mmol/L IDP
20u/mL Glycerol kinase(旭化成ファーマ株式会社、T-64: Flavobacterium meningosepticum由来)
ウサギ筋肉由来のピルビン酸キナーゼを用い、下記の組成の反応液を調製した。
50mmol/L PIPES-NaOH緩衝液(pH7.5)
5mmol/L 塩化マグネシウム(MgCl2)
10mmol/L グルコース
1mmol/L ADP-HKPII
1u/mL G6PDH
1mmol/L NAD
3mmol/L ATP
10u/mL ピルビン酸キナーゼ(PK、シグマ社、ウサギ筋肉由来)
下記の組成の反応液を調製した。
50mmol/L PIPES-NaOH緩衝液(pH7.0)
5mmol/L MgCl2
10mmol/L グリセロール
5u/mL パーオキシダーゼ (西洋わさび、Sigma)
0.03% 4-アミノアンチピリン
0.02% N-エチル-N-(2-ヒドロキシ-3-スルホプロピル)-3-メチルアニリン,ナトリウム塩,2水和物(TOOS、同仁化学)
1mmol/L GTP
1mmol/L ADP
20u/mL L-α-グリセロホスフェートオキシダーゼ (旭化成ファーマ株式会社、GPOSP、カタログNo.T-60)
5u/mL 3-ホスホグリセリン酸キナーゼ(PGK)(シグマ社製:酵母由来)
1u/mL グリセロキナーゼ(GK) (大腸菌由来、Sigma)
下記の組成の反応液を調製した。
50mmol/L PIPES緩衝液(pH7.0)
5mmol/L MgCl2
0.5mmol/L ADP
10mmol/L グリセロール
0.3% 4-アミノアンチピリン
20u/mL GPOSP(旭化成ファーマ株式会社、カタログNo.T-60)
0.2% TOOS (同仁化学)
4.5u/mL パーオキシダーゼ(シグマ)
1u/mL グリセロキナーゼ(大腸菌由来:シグマ)
50u/mL HKIII(旭化成ファーマ株式会社;カタログNo.T-141)
下記の組成の反応液を調製した。
50mmol/L PIPES緩衝液(pH7.0)
5mmol/L MgCl2
5mmol/L クレアチンリン酸
0.5mmol/L AMP
1mmol/L CDP
0.5mmol/L deamido-NAD
20mmol/L NH4Cl
1mmol/L コール酸ナトリウム
0.5u/mL NAD合成酵素(NADSII:旭化成ファーマ株式会社、T-67)
2u/mL 12α-ヒドロキシステロイドデヒドロゲナーゼ(12α-HSDII:旭化成ファーマ株式会社、T-190)
10u/mL CK(HC-CKII: 旭化成ファーマ株式会社、T-74)
下記の組成の反応液を調製した。
50mmol/L Tris-HCl緩衝液(pH9.0)
5mmol/L 塩化マグネシウム
10mmol/L グルコース
50mmol/L 塩化カリウム
1u/mL ADP-HK(旭化成ファーマ株式会社製:T-92)
1u/mL G6PDH(東洋紡製)
1mmol/L NAD
2mmol/L ATP
50u/mL ピルビン酸キナーゼ(PK、シグマ社、ウサギ筋肉由来)
マウスB型アイソザイムをコードするDNAを常法により取得した。CK(旭化成ファーマ株式会社製、カタログNo.T-74)生産に用いられている宿主、ベクター系にこの遺伝子を挿入してクローニングし、常法に従いCK-B(ヒト由来B型アイソザイム)を取得した。
50mmol/L TES緩衝液(pH7.5)
12mmol/L 塩化マグネシウム
12mmol/L グルコース
1.5u/mL ADP-HK(旭化成ファーマ株式会社製:T-92)
1.5u/mL G6PDH(東洋紡製)
1.5mmol/L チオNAD(オリエンタル酵母工業製)
0.7mmol/L ATP
50mmol/L TES緩衝液(pH7.5)
8mmol/L IDP
0.5mmol/L N-アセチルシステイン
0.02% BSA
16u/mL クレアチニンアミドハイドロラーゼ(東洋紡製)
200u/mL CK-B(ヒト由来B型アイソザイム)
Claims (32)
- キナーゼの正反応基質、そのリン酸化物、及びそれらへ誘導される誘導前物質の少なくとも一つの測定方法であって、
(1)キナーゼであって、ヌクレオチド補酵素の存在下、キナーゼの正反応基質からそのリン酸化物を生成する正反応、及びその逆反応を触媒し、前記正反応と前記逆反応において、少なくともそれぞれ別異のヌクレオシド部分を有するヌクレオチド補酵素を利用するキナーゼと、前記キナーゼの第一のヌクレオチド補酵素と、前記第一のヌクレオチド補酵素とはヌクレオシド部分が異なる第二のヌクレオチド補酵素と、を
試料、又は測定対象が誘導前物質である場合には前記誘導前物質を前記キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理を行った試料と接触せしめ、下記式(1)のサイクリング反応を反応せしめる工程と、
(2)前記第一のヌクレオチド補酵素、前記第一のヌクレオチド補酵素の変化物、前記第二のヌクレオチド補酵素、及び前記第二のヌクレオチド補酵素の変化物の少なくともいずれかの変化量に対応するシグナルの変化量を検出する工程と、
(3)前記検出されたシグナルの変化量に基づき、前記試料が含有するキナーゼの正反応基質、そのリン酸化物、及びそれらへ誘導される誘導前物質の少なくとも一つの量を算出する工程と、
を含む測定方法。 - 測定対象が前記キナーゼの正反応基質及び/又はそのリン酸化物である、請求項1に記載の測定方法。
- 測定対象が前記誘導前物質であり、前記試料が、前記誘導前物質を、前記キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理を行った試料であり、
前記検出されたシグナルの変化量に基づき、前記誘導前物質の量が算出される、
請求項1に記載の測定方法。 - 測定対象が前記誘導前物質であるクレアチニン、前記キナーゼがクレアチンキナーゼ、前記キナーゼの正反応基質がクレアチンである、請求項1、4及び5のいずれかに記載の測定方法。
- 前記誘導前物質を、前記キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理において、クレアチニンに、水の存在下、クレアチニンアミドヒドロラーゼ(EC3.5.2.10)を接触させることを含む、請求項1、及び4ないし6のいずれかに記載の測定方法。
- 前記第一のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシンキサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン、及びデオキシイノシンのいずれかである、請求項1ないし7のいずれかに記載の測定方法。
- 前記第二のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン及びデオキシイノシンのいずれかである、請求項1ないし7のいずれかに記載の測定方法。
- 前記第一のヌクレオチド補酵素のヌクレオシド部分と前記第二のヌクレオチド補酵素のヌクレオシド部分の組み合わせが、アデノシンとイノシン、グアノシンとアデノシン、デオキシアデノシンとグアノシン、デオキシアデノシンとデオキシグアノシン、デオキシアデノシンとイノシン、又はイノシンとアデノシンである、請求項1ないし9のいずれかに記載の測定方法。
- 前記第一のヌクレオチド補酵素のヌクレオシド部分がアデノシンであり、前記第二のヌクレオチド補酵素のヌクレオシド部分がイノシンである、請求項1ないし10のいずれかに記載の測定方法。
- 前記第一のヌクレオチド補酵素がアデノシン3リン酸(ATP)であり、前記第二のヌクレオチド補酵素がイノシン2リン酸(IDP)である、請求項1ないし請求項11のいずれかに記載の測定方法。
- 前記シグナルの変化量を検出する工程において、正反応による前記第一のヌクレオチド補酵素の変化物を利用できるが、前記第二のヌクレオチド補酵素を利用できない検出用酵素、又は前記第一のヌクレオチド補酵素を利用できないが、逆反応による前記第二のヌクレオチド補酵素の変化物を利用できる検出用酵素を用いて、前記正反応による第一のヌクレオチド補酵素の変化物又は前記逆反応による第二のヌクレオチド補酵素の変化物の増加量に対応する前記シグナルの変化量を検出する、請求項1ないし12のいずれかに記載の測定方法。
- 前記シグナルの変化量を検出する工程において、アデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 2.7.1.147)を用いて、グルコースの存在下、アデノシン2リン酸(ADP)の増加量に対応して変化するシグナルの変化量を検出する、請求項1ないし13のいずれかに記載の測定方法。
- 前記シグナルの変化量を検出する工程において、アデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 2.7.1.147)を用いて、チオニコチンアミドアデニンジヌクレオチドリン酸(チオNADP)、チオニコチンアミドアデニンジヌクレオチド(チオNAD)、ニコチンアミドアデニンジヌクレオチドリン酸(NADP)、及びニコチンアミドアデニンジヌクレオチド(NAD)のいずれかの補酵素と、グルコースと、グルコース6リン酸デヒドロゲナーゼと、の存在下、アデノシン2リン酸(ADP)の増加量に対応して変化するシグナルの変化量を検出する、請求項1ないし14のいずれかに記載の測定方法。
- キナーゼの正反応基質、そのリン酸化物、及びそれらへ誘導される誘導前物質の少なくとも一つを測定するための測定用組成物であって、測定対象が誘導前物質である場合には、前記誘導前物質を前記キナーゼ正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理をした後に用いられ、且つ
(a)キナーゼであって、ヌクレオチド補酵素の存在下、当該キナーゼの正反応基質からそのリン酸化物を生成する正反応、及びその逆反応を触媒し、前記正反応と前記逆反応において、少なくともそれぞれ別異のヌクレオシド部分を有するヌクレオチド補酵素を利用するキナーゼと、
(b)前記正反応における第一のヌクレオチド補酵素と、
(c)前記第一のヌクレオチド補酵素とはヌクレオシド部分が異なる、前記逆反応における第二のヌクレオチド補酵素と、
を備える、組成物。 - 測定対象が前記キナーゼの正反応基質及び/又はそのリン酸化物である、請求項16に記載の組成物。
- 測定対象が誘導前物質であり、当該誘導前物質を、前記キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理をした後に用いられる、請求項16に記載の組成物。
- 測定対象が前記誘導前物質であるクレアチニン、前記キナーゼがクレアチンキナーゼ、前記キナーゼの正反応基質がクレアチンである、請求項16、19及び20のいずれかに記載の組成物。
- 前記誘導前物質を、前記キナーゼの正反応基質及び/又はそのリン酸化物へと定量的に誘導せしめる処理において、クレアチニンに、水の存在下、クレアチニンアミドヒドロラーゼ(EC3.5.2.10)を接触させ、クレアチンを誘導せしめる、請求項16、及び19ないし21のいずれかに記載の組成物。
- 前記第一のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン、及びデオキシイノシンのいずれかである、請求項16ないし22のいずれかに記載の組成物。
- 前記第二のヌクレオチド補酵素のヌクレオシド部分が、アデノシン、グアノシン、チミジン、ウリジン、シチジン、キサントシン、イノシン、デオキシアデノシン、デオキシグアノシン、デオキシチミジン、デオキシウリジン、デオキシシチジン、デオキシキサントシン、及びデオキシイノシンのいずれかである、請求項16ないし22のいずれかに記載の組成物。
- 前記第一のヌクレオチド補酵素のヌクレオシド部分と前記第二のヌクレオチド補酵素のヌクレオシド部分の組み合わせが、アデノシンとイノシン、グアノシンとアデノシン、デオキシアデノシンとグアノシン、デオキシアデノシンとデオキシグアノシン、デオキシアデノシンとイノシン、又はイノシンとアデノシンである、請求項16ないし24のいずれかに記載の組成物。
- 前記第一のヌクレオチド補酵素のヌクレオシド部分がアデノシンであり、前記第二のヌクレオチド補酵素のヌクレオシド部分がイノシンである、請求項16ないし25のいずれかに記載の組成物。
- 前記第一のヌクレオチド補酵素がアデノシン3リン酸(ATP)であり、前記第二のヌクレオチド補酵素がイノシン2リン酸(IDP)である、請求項16ないし26のいずれかに記載の組成物。
- 正反応による前記第一のヌクレオチド補酵素の変化物を利用できるが、前記第二のヌクレオチド補酵素を利用できない検出用酵素、又は前記第一のヌクレオチド補酵素を利用できないが、逆反応による前記第二のヌクレオチド補酵素の変化物は利用できる検出用酵素を更に含む、請求項16ないし27のいずれかに記載の組成物。
- 前記検出用酵素がアデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 2.7.1.147)であり、グルコースを更に含む、請求項28に記載の組成物。
- チオニコチンアミドアデニンジヌクレオチドリン酸(チオNADP)、チオニコチンアミドアデニンジヌクレオチド(チオNAD)、ニコチンアミドアデニンジヌクレオチドリン酸(NADP)又はニコチンアミドアデニンジヌクレオチド(NAD)と、
グルコース6リン酸デヒドロゲナーゼと、
を更に含む、請求項28に記載の組成物。 - 測定対象が前記誘導前物質であるクレアチニンであり、
当該クレアチニンに、水の存在下、クレアチニン アミドヒドロラーゼ(EC3.5.2.10)を接触させ、クレアチニンをクレアチンへと定量的に誘導せしめる処理をした後に用いられ、
(a)クレアチンキナーゼと、
(b)アデノシン3リン酸(ATP)と、
(c)イノシン2リン酸(IDP)と、
(d)アデノシン2リン酸(ADP)依存性グルコキナーゼ(EC 2.7.1.147)と、
(e)グルコースと、
(f)チオニコチンアミドアデニンジヌクレオチドリン酸(チオNADP)、チオニコチンアミドアデニンジヌクレオチド(チオNAD)、ニコチンアミドアデニンジヌクレオチドリン酸(NADP)又はニコチンアミドアデニンジヌクレオチド(NAD)と、
(g)グルコース6リン酸デヒドロゲナーゼと、
を備える、請求項16、及び19ないし22のいずれかに記載の組成物。 - 請求項16ないし31のいずれかに記載の組成物を含む、試薬キット。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016550299A JP6450394B2 (ja) | 2014-09-26 | 2015-09-18 | キナーゼを用いた新規な測定方法及び組成物 |
CN201580049107.8A CN107075558B (zh) | 2014-09-26 | 2015-09-18 | 使用激酶的新型测定方法及组合物 |
EP15844093.3A EP3199639B1 (en) | 2014-09-26 | 2015-09-18 | Novel measurement method using kinase, and composition |
US15/510,059 US10472665B2 (en) | 2014-09-26 | 2015-09-18 | Measuring method and composition using kinase |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014-196720 | 2014-09-26 | ||
JP2014196720 | 2014-09-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016047580A1 true WO2016047580A1 (ja) | 2016-03-31 |
Family
ID=55581107
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2015/076639 WO2016047580A1 (ja) | 2014-09-26 | 2015-09-18 | キナーゼを用いた新規な測定方法及び組成物 |
Country Status (5)
Country | Link |
---|---|
US (1) | US10472665B2 (ja) |
EP (1) | EP3199639B1 (ja) |
JP (1) | JP6450394B2 (ja) |
CN (1) | CN107075558B (ja) |
WO (1) | WO2016047580A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020080249A1 (ja) * | 2018-10-19 | 2020-04-23 | 旭化成ファーマ株式会社 | 酵素的測定方法及び酵素的測定用試薬 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110779887B (zh) * | 2019-09-30 | 2021-05-14 | 浙江大学 | 一种测定磷酸甘油酸激酶活性的方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6047698A (ja) * | 1983-08-26 | 1985-03-15 | Toyo Jozo Co Ltd | Νh↓3またはatpの測定法 |
JP2002355095A (ja) * | 2000-12-05 | 2002-12-10 | Toyobo Co Ltd | 生体成分の測定方法およびそれに用いる試薬キット |
JP2006223163A (ja) * | 2005-02-16 | 2006-08-31 | Hiroshima Univ | Atp増幅−サイクリング法による微量atpの定量方法およびキット |
WO2010082665A1 (ja) * | 2009-01-19 | 2010-07-22 | 旭化成ファーマ株式会社 | メバロン酸、3-ハイドロキシメチルグルタリルコエンザイムaおよびコエンザイムaの測定方法ならびに測定試薬 |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0673479B2 (ja) | 1989-12-01 | 1994-09-21 | 旭化成工業株式会社 | 胆汁酸の高感度定量法および定量用組成物 |
JP2713783B2 (ja) | 1989-12-11 | 1998-02-16 | 株式会社トクヤマ | 高感度測定法 |
JP3036708B2 (ja) | 1991-05-14 | 2000-04-24 | 旭化成工業株式会社 | D−グルコース−6−リン酸の高感度定量法および定量用組成物 |
JP3155415B2 (ja) | 1993-12-03 | 2001-04-09 | 株式会社トクヤマ | ベンジルアミン類の高感度定量方法 |
JP3151097B2 (ja) | 1993-12-24 | 2001-04-03 | 株式会社トクヤマ | チラミンの高感度定量方法 |
DE69637583D1 (de) * | 1995-12-27 | 2008-08-14 | Asahi Kasei Pharma Corp | Verfahren zur bestimmung von vitalproben |
JPH09187295A (ja) * | 1996-01-09 | 1997-07-22 | Hitachi Ltd | 酵素活性測定方法およびその装置 |
JP4155415B2 (ja) | 1996-02-19 | 2008-09-24 | 旭化成ファーマ株式会社 | 生体試料の測定法 |
EP1264894A4 (en) * | 2000-01-17 | 2005-11-30 | Satake Eng Co Ltd | REACTION SYSTEMS FOR ATP REGENERATION AND METHOD FOR THE CONTROL OF ADENIN NUCLEOTIDES, METHOD FOR THE DETECTION OF RNA AND METHOD FOR THE AMPLIFICATION OF ATP USING THEREOF |
US6867012B2 (en) | 2000-12-05 | 2005-03-15 | Toyo Boseki Kabushiki Kaisha | Determination method of biological component and reagent kit used therefor |
US7338775B1 (en) * | 2005-02-09 | 2008-03-04 | Myriad Genetics, Inc. | Enzyme assay and use thereof |
JP4723311B2 (ja) * | 2005-08-17 | 2011-07-13 | シスメックス株式会社 | クレアチンキナーゼ活性測定用試薬 |
WO2011056611A1 (en) * | 2009-10-26 | 2011-05-12 | Myrexis, Inc. | Coupled reactions for analysis of nucleotides and their hydrolysis |
JP6522466B2 (ja) * | 2015-08-20 | 2019-05-29 | 旭化成ファーマ株式会社 | オートタキシンの活性測定方法 |
-
2015
- 2015-09-18 CN CN201580049107.8A patent/CN107075558B/zh active Active
- 2015-09-18 JP JP2016550299A patent/JP6450394B2/ja active Active
- 2015-09-18 EP EP15844093.3A patent/EP3199639B1/en active Active
- 2015-09-18 US US15/510,059 patent/US10472665B2/en active Active
- 2015-09-18 WO PCT/JP2015/076639 patent/WO2016047580A1/ja active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6047698A (ja) * | 1983-08-26 | 1985-03-15 | Toyo Jozo Co Ltd | Νh↓3またはatpの測定法 |
JP2002355095A (ja) * | 2000-12-05 | 2002-12-10 | Toyobo Co Ltd | 生体成分の測定方法およびそれに用いる試薬キット |
JP2006223163A (ja) * | 2005-02-16 | 2006-08-31 | Hiroshima Univ | Atp増幅−サイクリング法による微量atpの定量方法およびキット |
WO2010082665A1 (ja) * | 2009-01-19 | 2010-07-22 | 旭化成ファーマ株式会社 | メバロン酸、3-ハイドロキシメチルグルタリルコエンザイムaおよびコエンザイムaの測定方法ならびに測定試薬 |
Non-Patent Citations (3)
Title |
---|
ETSURO ITO ET AL.: "Development of Super High- Sensitive Measurement of Proteins, Rinsho Byori", THE 57TH CHUGOKU-SHIKOKU REGIONAL CONGRESS SYMPOSIUM: SAISHIN NO RINSHO KENSA UP TO DATE(1), vol. 60, no. 11, 2012, pages 1088 - 1093, XP009501180 * |
MATSUOKA T ET AL.: "An ultrasensitive enzymatic method for measuring mevalonic acid in serum.", J. LIPID RES., vol. 53, no. 9, 2012, pages 1987 - 1992, XP009165622, DOI: doi:10.1194/jlr.D028621 * |
See also references of EP3199639A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020080249A1 (ja) * | 2018-10-19 | 2020-04-23 | 旭化成ファーマ株式会社 | 酵素的測定方法及び酵素的測定用試薬 |
JPWO2020080249A1 (ja) * | 2018-10-19 | 2021-10-07 | 旭化成ファーマ株式会社 | 酵素的測定方法及び酵素的測定用試薬 |
US11773379B2 (en) | 2018-10-19 | 2023-10-03 | Sysmex Corporation | Enzymes and reagents for measurement of short chain fatty acids |
JP7433238B2 (ja) | 2018-10-19 | 2024-02-19 | 旭化成ファーマ株式会社 | 酵素的測定方法及び酵素的測定用試薬 |
Also Published As
Publication number | Publication date |
---|---|
CN107075558A (zh) | 2017-08-18 |
CN107075558B (zh) | 2021-09-24 |
EP3199639B1 (en) | 2020-12-30 |
US10472665B2 (en) | 2019-11-12 |
US20170306389A1 (en) | 2017-10-26 |
JP6450394B2 (ja) | 2019-01-09 |
EP3199639A1 (en) | 2017-08-02 |
EP3199639A4 (en) | 2018-04-18 |
JPWO2016047580A1 (ja) | 2017-06-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6450394B2 (ja) | キナーゼを用いた新規な測定方法及び組成物 | |
JP5458487B2 (ja) | リン酸の測定方法 | |
JP6522466B2 (ja) | オートタキシンの活性測定方法 | |
JP6487711B2 (ja) | プリンヌクレオシドホスホリラーゼを用いた、オルトリン酸、アルカリホスファターゼ、及びピロリン酸等の新規な測定方法、並びに組成物 | |
US10731200B2 (en) | Quantification method for ammonia, quantification reagent kit, test piece, and ammonia quantification device | |
CN109385409B (zh) | 谷氨酰胺合成酶反应方法、氨定量方法、谷氨酰胺合成酶反应用试剂和氨定量用试剂盒 | |
JP6989300B2 (ja) | アンモニアの定量方法、定量試薬キット、試験片及びアンモニアの定量装置 | |
JP2006187251A (ja) | ピロリン酸の定量方法 | |
Escobedo-Hinojosa et al. | A real-time 31P-NMR-based approach for the assessment of glycerol kinase catalyzed monophosphorylations | |
JP2009183203A (ja) | ホスファターゼの測定方法 | |
JP3034987B2 (ja) | D−グリセロアルデヒド−3−リン酸、無機リン、または1,3−ジホスホグリセリン酸の高感度定量法および定量用組成物 | |
Hu et al. | A bioluminescent method for measuring thymidylate kinase activity suitable for high-throughput screening of inhibitor | |
JP2818696B2 (ja) | Nadhキナーゼを用いる高感度定量法 | |
JPH047200B2 (ja) | ||
JP5633669B2 (ja) | Adpの測定方法およびadp測定用キット | |
WO2023074687A1 (ja) | ピロリン酸又はホスホリボシルピロリン酸の測定方法、これらを測定するための測定用組成物及び試薬キット並びにこれらの増幅方法 | |
WO2021193489A1 (ja) | 遊離脂肪酸濃度測定方法および遊離脂肪酸濃度測定用試薬チップ | |
JP2001252095A (ja) | 微量成分の測定法及びそれに用いる組成物 | |
JPH04335899A (ja) | L−グリセロール−3−リン酸またはジヒドロキシアセトンリン酸の高感度定量法および定量用組成物 | |
JP2002186497A (ja) | 1,5−アンヒドログルシトールの定量方法 | |
JPH11195A (ja) | クレアチンキナーゼまたはそのmbアイソザイムの定量用乾式分析素子 | |
JP3750956B2 (ja) | クレアチンキナーゼmbアイソザイムの定量用乾式分析素子 | |
JP3936976B2 (ja) | Ampの酵素的分解 | |
Bao et al. | Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP | |
WO2012036675A1 (en) | Phosphatase coupled kinase assay |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15844093 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2016550299 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15510059 Country of ref document: US |
|
REEP | Request for entry into the european phase |
Ref document number: 2015844093 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2015844093 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |