JPH027640B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention utilizes components in a test solution, such as pre-existing ATP (adenosine triphosphate),
Or relates to the determination of ATP released and produced by the reaction of ADP (adenosine diphosphate) and a phosphorus compound for a kinase substrate, or a method for measuring any one component of a kinase, ADP, or a phosphorus compound for a kinase substrate based on the quantification of ATP. . Conventionally, creatine and ATP are released from free or generated ATP, such as creatine phosphate and ADP, by the action of creatine kinase.
ATP in the generated reaction system and pre-existing
As a method for measuring ATP, for example, glucose and hexokinase are applied to this ATP.
ADP and glucose-6-phosphate are produced, and NADP (nicotine adenine dinucleotide phosphate) and glucose-6-phosphate dehydrogenase are made to act on the glucose-6-phosphate to produce glucono-δ-lactone and reduced form
to generate NADP, and then this reduced NADP
The amount of produced was determined by absorbance measurement at wavelengths in the ultraviolet region. (Methods in Engymatic
Analysis Vol. 2, p. 789). In this way, with regard to conventional ATP measurements or ATP measurements in reaction systems that release and generate ATP, only reduced NADP, which is a quantitative component, is produced from 1 molar ratio of ATP, and is still insufficient in terms of quantitative sensitivity. It was something. The present inventors have conducted various studies on methods that can quantify even minute amounts of ATP with good sensitivity. As a result, we have found that at least the ATP present in the test liquid contains fatty acids and CoASH (co-enzyme). A) is subjected to the action of acyl-CoA synthetase (EC6.2.1.8) to generate acyl-CoA, and the generated acyl-CoA is converted into acyl-CoA.
Dehydroacyl-CoA is generated by the action of oxidase (see US Pat. No. 4,346,173), and this dehydroacyl-CoA is further converted to hydroxyacyl-CoA by the action of enoyl-CoA hydratase (EC4.2.1.17). This hydroxyacyl-CoA is converted into 3 using NAD (nicotine adenine dinucleotide).
-Hydroxyacyl-CoA dehydrogenase (EC1.1.1.35) generates 3-ketoacyl-CoA with the production of reduced NAD, and further converts this 3-ketoacyl-CoA into CoASH.
is used to generate acyl-CoA with the production of acetyl-CoA through the action of 3-ketoacyl-CoA thiolase (EC2.3.1.16), and this generated acyl-CoA is further injected into the above-mentioned acyl-CoA.・Acts as a substrate for oxidase, enters this reaction cycle, and in each cycle, acyl-CoA produces acyl-CoA whose number of carbon atoms is reduced by two units, and at this time, the components of the change that can be detected in the reaction system, For example, the amount of reduced NAD produced from NAD is an equimolar amount per cycle of acyl-CoA, and in this way, the amount of reduced NAD produced from acyl-CoA formed by 1 molar ratio of ATP is reduced to the number of cycles. It is made by producing a high molar ratio of reduced NAD according to the
We have completed a method to quantify ATP with high sensitivity. The present invention was completed based on the above findings, and in quantifying ATP in a test solution, the following reaction steps are performed: , a reaction step in which a test solution containing ATP is reacted with a fatty acid, CoASH, and an enzyme having acyl-CoA synthetase activity to form acyl-CoA;
A reaction step for converting dehydroacyl-CoA into hydrogen peroxide and dehydroacyl-CoA using an enzyme having CoA/oxidase activity, a reaction step for converting dehydroacyl-CoA to hydroxyacyl-CoA using water and an enzyme having enoyl-OcA/hydratase activity, hydroxyacyl -CoA, NAD and 3
- A reaction step for converting ketoacyl-CoA into reduced NAD and ketoacyl-CoA with an enzyme having hydroxyacyl-CoA/dehydrogenase activity, a reaction step for converting ketoacyl-CoA into acyl-CoA using an enzyme having CoASH and 3-ketoacyl-CoA/thiolase activities. In the reaction step including reagents and enzymes for the reaction, the acyl-CoA produced in the step is subjected to a cycling reaction as a substrate for the acyl-CoA in the step, and at least 2 Cycling the reaction for more than one cycle, then measuring at least one component of the amount of the consumed component or the amount of the component produced in a high molar ratio that can be detected depending on the number of cycles of the cycling reaction, and optionally step As an equivalent step, acyl-CoA was used instead of an enzyme with acyl-CoA oxidase activity in
dehydroacyl by an electron acceptor and an enzyme with acyl-CoA dehydrodenase activity
This is a quantitative method characterized by a reaction step including a step of converting CoA. First, the test solution of the present invention is at least ATP.
Any test liquid containing ATP may be used, and examples include test liquids that contain ATP in advance and ATP-containing test liquids that release ATP. As an ATP-containing test solution that has released ATP, the ADP is usually phosphorylated through an enzymatic reaction with a kinase, ADP, and a phosphorus compound for the kinase substrate.
Examples include enzyme reaction systems that liberate and produce . More specifically, the following enzymatic reaction systems are exemplified, but these are merely illustrative and do not limit the scope of the present invention in any way. Creatine phosphate + ADP reducing agent, Mg ²⁺ ―――――――――――――――――→ Creatine kinase (EC2.7.3.2) Creatine + ATP (Reducing agent: β-mercaptoethanol, reduced form glutathione, cysteine, N-acetylcysteine, dithiothreitol, etc.) Phosphoenolpyruvate + ADPMg ²⁺ or Mn ²⁺ and K
⁺ , NH ⁺ ₄ Rb ⁺ or ――――――――――――――――――→ Pyruvate kinase (EC2.7.1.40) Pyruvate + ATP Acetyl phosphate + ADPMg ²⁺ or Mn ²⁺ ――――――――――――――――→ Acetate kinase (EC2.7.2.1) Acetic acid + ATP Carbamoyl phosphate + ADPMg ²⁺ ―――――――――――― ――――――→ Carbamate kinase (EC2.7.2.2) NH ₃ +CO ₂ +ATP 4-phospho-L-aspartate + ADPMg ²⁺ ―――――――――――――――― ――→ Aspartate kinase (EC2.7.2.4) L-aspartate + ATP 1.3-diphospho-D-glycerate + ADPMg ²⁺ or Mn ²⁺ ―――――――――――――――― ――――→ Phosphoglycerate kinase (EC2.7.2.3) 3-phospho-D-glycerate + ATP Arginine phosphate + ADPMg ²⁺ or Mn ²⁺ ―――――――――― ―――――― ---→ Arginine kinase (EC2.7.3.3) L-arginine +
ATP ADP＋ADPMg ²⁺ ――――――――――――――→ Myokinase (EC2.7.4.3) AMP＋ATP XDP＋ADPMg ²⁺ , Mn ²⁺ or Ca ²⁺ XDP＋ADPMg ²⁺ , Mn ²⁺ or Ca ^{2 +} ――――――――――――――――――――――――
―――→ XDP＋ADPMg ²⁺ , Mn ²⁺ or Ca ²⁺ ――――――――――――――――――――――――
――→ Nucleoside monophosphate kinase (EC2.7.4.4
) XMP+ATP (X=U, I, G, C or A) Kinases in these enzyme reaction systems include, for example, the above-mentioned creatine phosphate, phosphoenolpyruvate, acetyl phosphate,
Carbamoyl phosphate, 4-phosphoaspartate, 1,3-diphospho-D-glycerate,
Any enzyme may be used as long as it has the function of generating and releasing ATP by transferring the phosphate group of the phosphorus compound for kinase substrates to ADP from phosphorus compounds for kinase substrates such as arginine phosphate, ADP, and XDP and ADP. For example, creatine kinase using creatine phosphate as the phosphorus compound for the kinase substrate, pyruvate kinase using phosphoenolpyruvate as the phosphorus compound for the kinase substrate, acetate kinase using acetyl phosphate as the phosphorus compound for the kinase substrate, and other carbamates. Examples include kinase, aspartate kinase, phosphoglycerate kinase, arginine kinase, myokinase, and nucleoside monophosphate kinase. In addition, the purpose of quantifying ATP in these enzyme reaction systems is to quantify any one of the following components: kinase activity measurement, ADP determination, and phosphorus compound determination for kinase substrates in the enzyme reaction system. , and the other two components may be used as reagents. In addition, in the enzyme reaction system that releases these ATP,
The amount of test solution and reagent used can be changed and designed as appropriate depending on the purpose of measurement and the conditions selected.
There are no particular limitations, and any conditions may be used as long as the amount of ATP released by the reaction is produced and released in a sufficient amount to quantify it. Furthermore, in order to release ATP, it is sufficient to carry out the reaction at a temperature around 37°C, and the reaction time may be longer than the time required to generate and release a sufficient amount of ATP.
It is done for more than a minute. Furthermore, as the fatty acid used in the present invention, saturated or unsaturated higher fatty acids having 10 or more carbon atoms are preferable. For example, capric acid with 10 carbon atoms, lauric acid with 12 carbon atoms, tridecylenic acid with 13 carbon atoms,
14 myristic acid, 15 carbon pentadecylenic acid, 16 carbon palmitic acid, palmitooleic acid, 17 carbon margaric acid, 18 carbon stearic acid, oleic acid, vaccenic acid, 12-octadecenoic acid, linoleic acid. Acid, linolenic acid, nonadecylene with 19 carbon atoms, arachidic acid with 20 carbon atoms, 9-icosenoic acid, 11-icosenoic acid, 11,14-icosadienoic acid, 8,11,14-icosatrienoic acid, arachidonic acid, carbon Examples include behenic acid with 21 carbon atoms, lignoceric acid with 22 carbon atoms, 11-docosenoic acid, and erucic acid. Although caprylic acid having 8 carbon atoms can also be used, it is preferable to use the above-mentioned higher fatty acids for measurement with high sensitivity. Furthermore, CoASH is used in the present invention. Next, the reaction steps of reacting ATP with a fatty acid to form acyl-CoA in order to measure ATP in a test solution of the present invention are exemplified as follows. Reaction step: This is a reaction step in which ATP is made to react with a fatty acid to form acyl-CoA. For example ATP
acyl in the coexistence of fatty acids and CoASH.
An acyl with enzymatic activity that catalyzes the reaction with CoA, AMP and pyrophosphate (PPi).
CoA/synthetase activity, fatty acids and
A reaction process based on CoASH may be mentioned. Reaction step: This is a reaction step for converting acyl-CoA to dehydroacyl-CoA. For example, acyl-CoA is converted into dehydroacyl-CoA in the presence of oxygen.
Examples include acyl-CoA oxidase activity and oxygen-based reaction steps that have enzymatic activity that catalyzes the reaction with CoA and hydrogen peroxide. Reaction step: This is a reaction step for converting dehydroacyl-CoA into hydroxyacyl-CoA. Examples include enoyl-CoA hydratase activity, which has an enzymatic activity that catalyzes the reaction of dehydroacyl-CoA to hydroxyacyl-CoA in the presence of water, and water-based reaction steps. Reaction step: This is a reaction step for converting hydroxyacyl-CoA into ketoacyl-CoA. For example, 3-hydroxyacyl-CoA dehydrogenase activity, which has an enzymatic activity that catalyzes the reaction of hydroxyacyl-CoA to ketoacyl-CoA and reduced NAD in the presence of NAD;
Mention may be made of reaction steps based on NAD. Reaction process: Ketoacyl-CoA to acyl-
This is a reaction process to make CoA. For example, 3 catalyzes the reaction of ketoacyl-CoA to acyl-CoA and acetyl-CoA in the presence of CoASH.
-ketoacyl-CoA thiolase activity and
A reaction process based on CoASH may be mentioned. In order to carry out each of these reaction steps, reagents required for each reaction and enzymes exhibiting enzymatic activity may be used, and the enzymes used include:
Enzymes derived from animals or microorganisms can be used, and commercially available enzymes may also be used.
Alternatively, it may be isolated from an enzyme-containing tissue. Examples of the enzyme (EC6.2.1.3) that exhibits acyl-CoA synthetase activity include those derived from guinea pig liver [J.Biol.Chem., 204 , 329 (1953)], those derived from rat, mouse, cow, and pig. Those derived from the liver (Japanese Unexamined Patent Publication No. 55-74791), those derived from Escherichia coli [Eur.J.
Biochem., 12 , 576 (1970)], those derived from Bacillus megaterium [Biochemistry 4 (1), 85 (1965)], and other producing bacteria belonging to the genus Aerobactor (A. aerogenes). IFO 3318), Seratia
Production bacteria belonging to the genus Seratia marcescens IFO
3054), Proteus mirabilis IFO 3849, Staphylococcus aureus IFO 3060, Pseudomonas aeruginosa IFO 3919), producing bacteria belonging to the genus Fusarium (Fusarium
oxysporum IFO 5942), Gibberella
Production bacteria belonging to the genus Gibberella fujikuroi IFO
6604), those derived from microorganisms such as production bacteria belonging to the genus Candida (Candida lipolytica IFO 0717) [J. Bacteriol., 105 (3) 1216 (1971),
J. Bacteriol., 114 (1) 249 (1973), Japanese Patent Publication No. 55-74790
JP-A No. 55-99187], etc. In addition, examples of enzymes that exhibit acyl-CoA oxidase activity include those derived from rat liver [Biochem.Biophy.Res.Commun., 83 (2) 479
(1978)], producing bacteria belonging to the genus Candida (Candida utilis, Candida lipolytica IFO
1548, Candida tropicalis IFO 0589), producing bacteria belonging to the genus Saccharomyces (Saccharomyces cerevisiae IFO 0213,
Saccharomyces cerevisiae Y0036:DSM2138,
FERM-P No.5174] Production bacteria belonging to the genus Eupenicillium (Eupenicillium javanicum IFO 7992), production bacteria belonging to the genus Monascus [Monascus sp.M-4800: DSM2136, FERM-
PNo.5225], a producing bacterium belonging to the genus Aspergillus [Aspergillus candidus M-
4815: DSM 2135, FERM-P No. 5226] Production bacterium belonging to the genus Arthrobacter [Arthrobacter sp.B-0720: DSM2137, FERM
- P No. 5224], production bacteria belonging to the genus Macrophomina (Macrophomina phaseoli ATCC14383), production bacteria belonging to the genus Cladosporium (Cladosporium resinae IFO 6367), etc. [Arch.Biochem.Biophys] .,
176, 591 (1976), JP-A-55-118391, JP-A-56-8683, JP-A-56-61991, U.S. Patent No. 4346173, West German Application No.
93223874, Specification No. 6]. In addition, instead of this acyl-CoA oxidase activity, acyl-CoA dehydrogenase [Acyl-CoA oxidase activity]
dehydrogenase, EC1.3.99.3, Acyl-CoA:
(acceptor) oxidoreductase] enzymes that exhibit activity, such as those derived from the liver of pigs, cows, and sheep [J.Biol.Chem., 218 , 717 (1956), J.Biol.Chem.
218, 701 (1956), J.Am.Chem.Soc., 75 , 2787
(1953), Biochim.Biophys.Acta., 22 , 475
(1956)], acyl-CoA may be converted into dehydroacyl-CoA. In this case, electron acceptors such as 2,6-dichlorophenol indophenol, 3-(p-iodophenyl)-2-(p-
Nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),
3,3'-(4,4'-biphenylylene)-bis(2,
5-diphenyl-2H-tetrazolium chloride) (Neo-TB), 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis[2-(p-
Nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (Nitrotetrazolium blue: NTB), 3,3'-(3,3'-dimethoxy-
4,4'biphenylylene)-bis[2,5-bis(p-nitrophenyl)-2H-tetrazolium.
Chloride] (TNTB), 3,3-(3,3'-dimethoxy-4,4'-biphenylene)-bis(2,5
-diphenyl-2H-tetrazolylam chloride (TB), etc., preferably together with phenazine methosulfate, may be used to carry out the reaction. Furthermore, an enzyme that exhibits enoyl-CoA/hydratase activity (EC4.2.1.17), an enzyme that exhibits 3-hydroxyalcy-CoA/dehydrogenase activity (EC1.1.1.35), and an enzyme that exhibits 3-ketoacyl-CoA/thiolase activity (EC2). .3.1.16) may be isolated from tissues containing enzyme activity as appropriate [J. Biol. Chem., 218 , 971 (1956), Angew.
Chem., 64 , 687 (1952), J.Biol.Chem., 207 ,
631 (1954), Biochim. Biophys. Acta., 26 , 448
(1957), J.Biol.Chem., 208 , 345 (1954)]. In addition, these enoyl-CoA hydratase activities,
For the 3-hydroxyacyl-CoA dehydrogenase activity and the 3-ketoacyl-CoA thiolase activity, it is preferable to use the enzyme activity of a multi-active enzyme having each enzyme activity on the same protein. Examples of this multi-active enzyme-producing bacterium include, for example, a producing bacterium belonging to the genus Escherichia.
coli; Proc. Natl. Acod. Sci., 74 (2) 492 (1977)],
Producing bacteria belonging to the genus Pseudomonas
Fragi B-0771 (FERM-PNo.5701); Patent application 1986
−99314 specification, US Patent Application No. 392010]
are mentioned. In particular, the production bacterium B-0771 belonging to the genus Pseudomonas and the multi-active enzyme obtained from this production bacterium are as follows.
First, this bacterium B-0771 was isolated from the soil of a pear field in Sutama-cho, Kitakoma-gun, Yamanashi Prefecture, and the findings of its cultivation on various media based on macroscopic and microscopic observations are as described below. (A) Macroscopic characteristics (1) Ordinary agar plate medium It is hill-shaped, circular, and forms a colony with a smooth periphery. It is semi-glossy and exhibits a grayish-white to pale yellow color.
No soluble pigments are produced. (2) Ordinary agar slant medium Grows well in a linear pattern. It is semi-gloss and has a grayish-white to pale yellow color. No soluble pigments are produced. (3) Liquid medium: It becomes cloudy and precipitates are formed. No bacterial membrane is formed. (B) Microscopic features Straight or slightly curved rods, singly or in pairs, sometimes in long chains. The size is
It is 0.4-0.6 x 0.5-3.0 μm and moves with polar hairs. Does not form spores. (C) Physiological and biochemical characteristics Gram staining - O/F test 0 Catalase + Oxidase + Lecithinase - Urease SSR medium + Christensen medium (+) Hydrolysis of gelatin - Hydrolysis of starch - Hydrolysis of casein - Aesculin - Hydrolysis of arginine + Poly-β-hydroxybutyrate (PHB)
- Production of indole - Production of hydrogen sulfide - Production of acetoin - MR test - Reduction of nitrate - Utilization of citric acid + Productivity of acid rather than sugar Acid production, no gas production: L (+) arabinose,
Cellobiose, fructose, fucose, galactose, glucose, glycerin, lactose,
Maltose, mannose, melibiose, rhamnose, sucrose, trehalose, xylose, non-acid-producing, non-gas producing: adonitol, dulcitol, mesoerythritol, inositol inulin, mannitol, melesitose, raffinose, salicin, sorbose, sorbitol, starch, as above , this bacterium B-0771 is Gram-negative,
It was recognized as a strain belonging to the genus Pseudomonas because it moves with polar hairs, is positive for catalase and oxidase, and is a bacterophilic bacterium that oxidatively decomposes glucose. In addition, The Journal of General
Microbiology (The Journal of General
Pseudomonas fragi described in Microbiology 25 , 379-408 (1961)
A comparison showed good agreement. [Table] Furthermore, an experiment was conducted to compare this bacterium B-0771 with the standard strain Pseudomonas fragi ATCC4973. The results were as shown in Table 1. [Table] [Table] As described above, this bacterium B-0771 was in good agreement with the standard strain Pseudomonas fragi ATCC4973. Therefore, this bacterium was classified as Pseudomonas fragi B-
0771 (Microorganism Accession Number Notification, Microorganism Accession No. 5701, FERM-P No.
5701”). Furthermore, we will discuss the method for measuring the activity of the multi-active enzyme isolated and purified by culturing this bacterium, and its physicochemical properties. (1) Activity measurement method (a) Enoyl-CoA/hydratase activity measurement method 0.2M Tris-HCl buffer (PH9.0) 0.4ml 1M KCl 0.1ml 40mM NAD 0.1ml 15mM Palmitoenoyl-CoA 0.1ml 1% Bovine serum albumin 0.05ml 0.25% NTB 0.1ml 30U/ml Diaphorase (manufactured by Toyo Jozo Co., Ltd.)
0.1ml 125U/ml・3-hydroxyacyl-CoA・
Dehydrogenase (manufactured by Boehringer)
0.01ml distilled water 0.04ml total 1.00ml The reaction solution having the above composition was prewarmed at 37℃ for 2 minutes, and 50μ of the enzyme solution was added to it.
Incubate at 37°C for 10 minutes. After the reaction,
The reaction is stopped by adding 2.0 ml of 0.5% sodium dodecyl sulfate, and then the absorbance (ΔA550) is measured at a wavelength of 550 nm. In the measurement, the amount of enzyme that produces 1 μmole of reduced NAD per minute is defined as 1 unit (1U). Furthermore, the enzyme activity follows the following formula. Enzyme activity (U/ml) = △A550×1/4×1000/50×1/10 (b) 3-Hydroxyacyl-CoA/dehydrogenase activity measurement method 0.2M/Tris-HCl buffer (PH8.5) 0.5ml 1M・KCl 0.05ml 40mM・NAD 0.1ml 15mM・3-hydroxypalmitoyl
CoA 0.1ml 1% bovine serum albumin 0.05ml 0.25% NTB 0.1ml 30U/ml Diaphorase 0.1ml Total 1.00ml Prewarm the reaction solution with the above composition at 37℃ for 2 minutes, and add 50μ of enzyme solution to this. In addition
Incubate at 37°C for 10 minutes. After the reaction,
The reaction is stopped by adding 2.0 ml of 0.5% sodium dodecyl sulfate, and then the absorbance (ΔA550) is measured at a wavelength of 550 nm. In the measurement, the amount of enzyme that produces 1 μmole of reduced NAD per minute is defined as 1 unit (1U). Furthermore, the enzyme activity follows the following formula. Enzyme activity (U/ml) = △A550×1/4×100/50×1/10 (c) 3-Ketoacyl-CoA Thiolase activity measurement method 0.2M Tris-HCl buffer (PH8.0) 0.2ml 10mM CoA SH 0.05ml 0.2mM 3-Ketopalmitoyl-CoA 0.1ml 100mM MgCl ₂ 0.1ml 1mM Dithiothreitol 0.1ml Distilled water 0.45ml Total 1.00ml Add the reaction solution having the above composition to a 1.0ml quartz cell. to 37°C, and add 20μ of enzyme solution to this.
was added to react at 37°C, and the absorbance (OD ₃₀₃ ) of the 3-ketopalmitoyl-CoA consumed by the reaction was measured over time at a wavelength of 303 nm. In the measurement, the amount of enzyme that consumes 1 μmole of 3-ketopalmitoyl-CoA per minute is defined as 1 unit (1 U). Furthermore, the enzyme activity follows the following formula. Enzyme activity (U/ml) = (OD per minute
₃₀₃ )×1/13.5×1000/20 (2) Enzyme action 1 mole of dehydroacyl-CoA and 1 mole of water to 1 mole of hydroxyacyl-CoA
Enoyl-CoA catalyzes the reaction that produces
Hydratase activity, a 3-hydroxyacyl- that catalyzes the reaction of 1 mole of hydroxyacyl-CoA and 1 mole of NAD to produce 1 mole of ketoacyl-CoA and 1 mole of reduced NAD.
CoA dehydrogenase activity, 1 mole ketoacyl-CoA and 1 mole CoA SH to 1
mole of acyl-CoA and 1 mole of acetyl-
Each enzyme activity of 3-ketoacyl-CoA thiolase activity that catalyzes the reaction that produces CoA is shown. (3) Proof that the three types of enzyme activities are on the same protein Purified enzyme is obtained through several purification steps from crude enzyme obtained from cells from a culture of Pseudomonas fragi B-0771. In this step, three types of enzyme activities were measured based on the above-mentioned activity measurement table (see below-mentioned production example of multi-active enzyme). The results were as shown in Table 2. [Table] (The enzyme activity measurement value is a value calculated by calculating the amount of protein in the enzyme-containing solution.) As a result, each enzyme activity is the ratio of the activity in each purification process from the crude enzyme. Since these three types of enzyme activities are in good agreement and the purified enzyme also has these three types of enzyme activities, it is recognized that these three types of enzyme activities are located on the same protein. Furthermore, purified enzyme obtained by Toyopearl HW-60 column chromatography in the production example described below.
After subjecting 2.0 mg to isoelectric focusing using carrier amphorite, the activities of the three enzymes were measured based on the activity measurement method. As a result, all three enzyme activities appeared on a single peak in the PH4.9 fraction. was detected. (4) Substrate specificity The following 3-hydroxyacyl-CoA having various carbon numbers were used as substrates, and their activities were measured according to the 3-hydroxyacyl-CoA dehydrogenase activity assay method. Substrate Relative activity (%) 3-hydroxycaproyl-CoA 56.5 3-hydroxycaprylyl-CoA 88.1 3-hydroxycapryl-CoA 99.0 3-hydroxylauryl-CoA 100.0 3-hydroxymyristoyl-CoA 98.0 3-hydroxypalmitoyl-CoA 75.5 3-hydroxystearyl-CoA 30.5 3-hydroxyalachideryl-CoA 9.5 3-hydroxyoleyl-CoA 57.5 3-hydroxylinolenyl-CoA 99.0 Furthermore, the present multi-active enzyme contains at least palmitoenoyl-CoA, 3-topalmitoyl-
It has substrate specificity for CoA. (5) 3-Hydroxypalmitoyl as an optimal PH substrate
Using CoA, 3-hydroxyacyl-CoA・
The activity was determined by changing the pH in the dehydrogenase activity measurement method using a Tris-HCl buffer solution pH 7.5 to 9.5. As a result, as shown in Figure 1, the optimum pH was around PH9. (6) PH stability 10mM various buffer solutions (PH4~7: dimethylglutaric acid-sodium hydroxide buffer, PH7.5~
The enzyme solution (15 U/ml) dissolved in Tris-HCl buffer (Tris-HCl buffer) was left to stand at 37°C for 60 minutes, and its residual activity was measured based on the 3-hydroxyacyl-CoA dehydrogenase activity assay method. As shown in FIG. 2, the results were stable in the pH range of 5 to 8. (7) Thermostability Enzyme solution (15U/ml) dissolved in 10mM dimethylglutaric acid-sodium hydroxide buffer (PH7.0)
After treatment at each temperature for 10 minutes, its residual activity was
-Hydroxyacyl-CoA dehydrogenase activity measurement method. As shown in Figure 3, the results were stable at temperatures up to approximately 50°C. (8) Isoelectric point The isoelectric point was measured at PH4.9 by focusing electrophoresis using carrier amphorite. (9) Effect of metal ions The effect on 3-hydroxyacyl-CoA dehydrogenase activity was measured. [Table] [Table] (10) Effect of surfactant The effect on 3-hydroxyacyl-CoA dehydrogenase activity was measured. [Table] As shown above, this multi-active enzyme is enoyl-
It has CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, and 3-ketoacyl-CoA thiolase activity on the same protein. Furthermore, the enzyme activity measuring method used for each of these enzyme activities is as follows. ●Acyl-CoA/syntheturbation activity measurement method 0.2M phosphate buffer (PH7.5) 0.2ml 10mM ATP 0.1ml 10mM MgCl ₂ 0.1ml 10mM CoASH 0.05ml 5% Triton X 1mM palmitic acid solution containing 100 ml 0.2ml distilled water 0.35ml total 1.00ml Prepare a first reaction solution having the above composition. In addition, a reaction solution having the following composition is prepared as the second reaction solution. 0.2M phosphate buffer (PH7.5) 0.5ml 20mM N-ethyl maleimide 0.1ml 15mM 4-aminoantipyrine 0.3ml 0.3% 3-methyl-N-ethyl-N-(β-hydroxyethyl)aniline 0.25ml peroxidase (100PPU/ml) 0.1ml 0.5% Sodium azide 0.1ml Acyl-CoA oxidase (120U/ml) 0.1ml Distilled water 0.55ml Total 2.00ml Add 50μ of the enzyme solution to the first reaction solution having the above composition and heat at 37°C. Allow to react for 10 minutes. After the reaction, this was added to the second reaction solution and allowed to react at 37°C for 5 minutes, and then the absorbance (ΔA ₅₅₀ ) was measured at a wavelength of 550 mm.
In addition, the enzyme activity follows the following formula. Enzyme activity (U/mg) = △A ₅₅₀ /10/32.0×1/2×3.
05/0.05×C (However, C indicates the concentration of acyl-CoA/synthetase in the enzyme solution (mg/ml).) Acyl-CoA/oxidase activity measurement method 0.2M Tris-HCl buffer (PH8.0) 0.1ml 5mM 4-aminoantipyrine 0.05ml 3mM Diethyl metatoluidine 0.05ml 0.5mg/ml peroxidase 0.05ml 25mM Palmitoyl-CoA 0.02ml Distilled water 0.23ml Total 0.50ml Add 10μ of the enzyme solution to the reaction solution having the above composition, After reacting at 37°C for 10 minutes, 0.5 ml of 4M urea was added to stop the reaction, and then 1% Triton Add 2 ml of 100 and compare the colors at a wavelength of 545 mm to determine the amount of hydrogen peroxide produced. Enzyme activity is defined as 1 unit (1U), which is the amount of enzyme that produces 1 μmole of hydrogen peroxide per minute. In addition, the enoyl-CoA/hydratase activity measurement method, the 3-hydroxyacyl-CoA/dehydrogenase activity measurement method, and the 3-ketoacyl-CoA/thiolase activity measurement method are the same as the methods described for each activity measurement method for the multi-active enzyme. This is due to Furthermore, in carrying out each reaction step, the amount of each enzyme activity used only needs to have sufficient enzyme activity to cause the reaction, and
It may be changed as appropriate depending on the amount of ATP, reaction conditions, etc., and is not particularly limited. for example
When reacting a test solution containing 0.005 to 0.05 μmole of ATP or producing or releasing it at 37°C for 30 seconds to 10 minutes, the acyl-CoA synthetase activity is usually 0.1 U or more, preferably 0.5 to
The amount of enzyme that exhibits an enzymatic activity of about 1 U may be used, and the amount of enzyme that exhibits an enzymatic activity of about 1 U or more for acyl-CoA oxidase activity, preferably about 5 to 15 U may be used. Furthermore, enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, 3
- Ketoacyl-CoA/thiolase activity, or the enzyme activity of a composite active enzyme having each of these enzyme activities on the same protein, is usually 0.1 U or more, preferably an amount of enzyme that exhibits an enzyme activity of about 1 to 25 U may be used. . Furthermore, reagents required for the reaction, such as fatty acids and CoASH required for the reaction process, NAD required for the reaction process, CoASH required for the reaction process
The amount of each reagent present in the test solution is
It is sufficient to use a sufficient amount equal to or more than the product of the amount of ATP and the number of β-oxidation cycles based on the number of carbon atoms of the fatty acid used. For example,
If ATP is 0.01μmole, fatty acid is usually 0.1μmole.
more than about 0.5μmole, preferably about 0.5μmole or more,
CoASH is about 0.1 μmole or more, preferably
About 0.5μmole or more, NAD about 0.1μmole or more,
Preferably, 0.5 μmole or more may be used. Furthermore, in the reaction process, in order to carry out the reaction based on acyl-CoA oxidase activity, oxygen existing in the reaction system, that is, dissolved oxygen, may be used, and water required for the reaction process may also be present in the reaction system. You can use water that is Furthermore, in order to improve the acyl-CoA synthetase activity, a water-soluble magnesium salt capable of releasing magnesium ions, preferably magnesium chloride, may be used. Further, in the reaction step, it is preferable to eliminate hydrogen peroxide generated based on acyl-CoA oxidase activity, and hydrogen peroxide may usually be decomposed and eliminated using catalase. In this way, at least fatty acids, CoASH, NAD, acyl-CoA synthetase activity, acyl-CoA oxidase activity,
Enoyl-CoA/hydratase activity, 3-hydroxyacyl-CoA/dehydrogenase activity,
What is necessary is to prepare a composition for measuring ATP containing each activity of 3-ketoacyl-CoA and thiolase activity, or at least two components of kinase, ADP, and a phosphorus compound for kinase substrate, fatty acid, CoASH, and NAD. , acyl-CoA・
Synthetase activity, acyl-CoA oxidase activity, enoyl-CoA hydratase activity,
A composition for quantitatively measuring any one component of kinase activity, ADP, and a phosphorus compound for kinase substrate in an enzyme reaction system containing each activity of 3-hydroxyacyl-CoA/dehydrogenase activity and 3-ketoacyl-CoA/thiolase activity. Just prepare it. In addition, in these compositions for measurement, the complex active enzyme is used for enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, and 3-ketoacyl-
It is preferably used in place of CoA/thiolase activity, and further preferably contains a water-soluble magnesium salt. Furthermore, it is preferable that this composition for measurement contains catalase and FAD (flavin adenine dinucleotide), and in addition,
Nonionic surfactants, such as Triton X-100
(trade name) to form a measurement composition, or generally neutral to weakly alkaline water or a buffer solution may be used to form a solution. Next, the measurement composition obtained in this way is used to measure ATP in various test solutions.First, the amount of test solution used is usually
It is sufficient to use 5μ or more and add it to usually 1ml or more of a solution of the composition for measurement, and the reaction conditions at that time are, for example, the reaction temperature is usually around 37°C. Further, the reaction time is not particularly limited, and a longer reaction time will produce a change in sensitivity, so it is usually 20 seconds or more, preferably about 30 seconds to 10 minutes. be.
In addition, the reaction medium is used to stabilize the activity of each enzyme.
Any medium in the PH range is sufficient, and is usually weakly acidic or weakly alkaline, such as phosphate buffer with pH 6.5 to 8.5, Tris-HCl buffer, imidazole-HCl buffer,
Dimethylglutaric acid-NaOH buffer and PIPES-NaOH buffer are used. After the reaction is carried out in this way, a detectable change in the reaction is measured, and this detectable change means that at least one molecule of the component is consumed or produced in one β-oxidation cycle. This is a change in Conveniently used in reactions
Reduced form produced from NAD through a reaction process
It is easy to detect and quantitatively measure changes in the amount of NAD. As a means of measuring this reduced NAD, for example, instead of using the absorption wavelength specific to the AND used,
The absorbance may be measured based on a wavelength in an absorption wavelength range that is an absorption wavelength specific to reduced NAD. N.A.D.
It has a specific maximum absorption wavelength around 260nm, and reduced NAD has a specific maximum absorption wavelength around 260nm and 340nm.
The absorption wavelength range, which is a specific absorption wavelength for measuring NAD, is around 320 nm to 360 nm, preferably around 340 nm. This wavelength measures the amount of reduced NAD produced as a detectable change. Further, as a means for measuring reduced NAD, there is also a method using a hydrogen atom transport system chromogen that has the ability to accept hydrogen atoms of reduced NAD. The hydrogen atom transport system chromogens that have the ability to accept the hydrogen atoms of reduced NAD include INT,
An electron acceptor such as a water-soluble tetrazolium salt such as MTT, Neo-TB, NTB, TNTB or TB may be used, and preferably one in which diaphorase is used together with a water-soluble tetrazolium salt to improve electron transfer. This hydrogen transport chromogen may be added to the measurement composition in advance, or may be added after the reaction,
The reduced NAD produced after the reaction reacts with this hydrogen atom transport system chromogen to cause a change in color, and this change in color tone can be measured by measuring the absorbance based on the absorption wavelength. Further, in detail about the reduced NAD produced, in the present invention,
In the β-oxidation cycle formed in the reaction process, the number of carbon atoms stops at 2 due to the substrate specificity of the catalytic enzyme to the carbon chain length of the fatty acid. Therefore carbon number is 16
In the case of palmitic acid and stearic acid, which are 1 or 18 saturated fatty acids, a 7 or 8 cycle reaction occurs, producing 7 or 8 moles of reduced NAD from 1 mole of palmitic acid or stearic acid. do. Furthermore, in the case of oleic acid having 18 carbon atoms and one unsaturated bond, 8 molar ratio of reduced NAD is produced. In this way, the number of cycle reactions in a saturated fatty acid or a fatty acid with one unsaturated bond is determined by calculating the difference in the number of carbon atoms (2) at which the reaction stops from the carbon chain length of the fatty acid, and then converting that value into the number of carbon atoms (2) in the β-oxidation cycle. This is the value obtained by calculating the quotient of 2 due to the disappearance of . Furthermore, in the case of linoleic acid, which is a fatty acid with 18 carbon atoms and two unsaturated bonds, 3-hydroxyacyl-
Due to the substrate specificity for stereoisomers in the action of CoA dehydrogenase activity, the reaction ends with the production of a 5 molar ratio of reduced NAD. Also 2
When using fatty acids having the above unsaturated bonds, in the presence of enoyl-CoA isomerase or 3-hydroxyacyl-CoA epimerase, the substrate specificity based on the stereoisomer does not affect the above. Similarly, reduced NAD is produced depending on the carbon chain length of the fatty acid. but,
When a fatty acid having two or more unsaturated bonds is used, the β-oxidation cycle is completed within 20 seconds, which is a very short time, and is therefore particularly suitable for ATP quantification. In this way, 5 for 1 mole of ATP.
Since it produces more than a mol of reduced NAD, it is possible to measure ATP or one component of the enzyme reaction system that releases ATP with high sensitivity.
Furthermore, as another method for measuring this reduced NAD, for example, diaphorase may be allowed to act on this reduced NAD in the presence of a fluorescent reagent such as resazurin, and the amount of fluorescent component produced by the reaction may be quantified. Furthermore, an enzyme that uses this reduced NAD as a substrate,
For example, reduced NAD/oxidase activity may be used to measure components that change based on this enzymatic reaction. Preferably, the enzyme is processed as an immobilized enzyme using a known immobilization method, and the immobilized enzyme is mounted on an oxygen electrode or the like. By using a reduced NAD/oxidase enzyme electrode, it is also possible to use a method for measuring reduced NAD by electrically measuring the amount of oxygen consumed by acting on the reduced NAD generated in the reaction system. Various known reduced forms
A means of quantifying NAD is used. Furthermore, the ATP content is calculated based on the measured amount of reduced NAD, and from this ATP content, quantitative values of kinase activity, ADP, and phosphorus compounds for kinase substrates in the enzyme reaction system in the test solution are calculated. Calculated. Furthermore, instead of measuring the reduced NAD described above, the amount of oxygen consumed in the reaction or the amount of hydrogen peroxide produced can be measured as a detectable change, for example, by an electrical change using an oxygen electrode or a hydrogen peroxide electrode. It's okay. In this way, the measurement method of the present invention and the composition based on the measurement method can be easily and extremely sensitively determined.
It is a good device that can measure ATP and can also be used to measure components in various test solutions that release ATP. ate, enolphosphopyruvate, acetyl phosphate,
In quantifying ADP, it can be easily and accurately measured with high sensitivity. Next, the present invention will be specifically described with reference to Examples and enzyme production examples, but the present invention is not limited by these in any way. Example 1 [Selection of fatty acids] 0.5M imidazole-NCl buffer (PH7.0) 0.1ml 40mM NAD 0.05ml 40mM CoASH 0.05ml 50mM MgCl ₂ 0.03ml 10mM ATP 0.1ml 1mM FAD 0.01ml Acyl - CoA/synthetase activity containing solution (Toyo Jozo Co., Ltd., 60 U/ml) 0.02 ml Acyl-CoA/oxidase activity containing solution (Toyo Jozo Co., Ltd., 150 U/ml) 0.1 ml Complex enzyme activity containing solution (enoyl-CoA/ Hydratase activity 400U/ml, 3-hydroxyacyl-CoA dehydrogenase activity 200U/ml, 3
-Ketoacyl-CoA/thiolase activity 163U/
ml; manufactured by Toyo Jozo Co., Ltd.; refer to the production example of multi-active enzymes described below) 0.1 ml catalase (manufactured by Boehringer Company, 260000 U/ml)
0.02ml Purified water 0.42mlTotal 1.0ml To 1.0ml of the reaction solution having the above composition, 20μ of various fatty acid containing solutions of 0.5mM palmitic acid, oleic acid, and linoleic acid were added and reacted at 37°C. Reduced NAD produced in liquid
The amount of was measured as the increase in absorbance at 340 nm over time. The results are shown in Figure 4.
In the figure, the solid line (--) is for linoleic acid, the dotted line (---) is for palmitic acid, and the dashed line (--
--) indicates the case of oleic acid. As a result, in the case of palmitic acid and oleic acid,
The reaction progresses quickly up to an increase of about 60-70%, but
It took 3-4 minutes to reach the end point. In addition, in the case of linoleic acid, the end point was reached in a very short time, within about 20 seconds, and this result suggests that
It was determined that using linoleic acid to convert the ATP-containing test solution into reduced NAD is particularly preferable because it can be done efficiently in a short time, and that the reaction time of about 30 seconds is sufficient for measurement. It was done. [Quantification of ATP using linoleic acid] A composition for measuring ATP was prepared by using linoleic acid in place of ATP in the above reaction solution. That is, a reaction solution having the following composition was prepared. 0.5M imidazole-HCl buffer (PH7.0) 0.1ml 40mM NAD 0.05ml 40mM CoASH 0.05ml 50mM MgCl ₂ 0.03ml 10mM linoleic acid (dissolved in 2% TritonX-100)
0.1ml 1mM・FAD 0.01ml Acyl-CoA・Synthase activity containing solution (60U/
ml) 0.02ml Solution containing acyl-CoA/oxidase activity (150U/ml) 0.1ml Solution containing complex active enzyme (enoyl-CoA/hydratase activity 400U/ml, 3-hydroxyacyl-
CoA/dehydrogenase activity 200U/ml, 3-ketoacyl-CoA/thiolase activity 163U/ml)
0.1ml Catalase (260000U/ml) 0.02ml Purified water 0.42mlTotal 1.0ml To 1.0ml of this reaction solution, add 20μ of ATP solutions of various concentrations with ATP content of 0 to 0.4mM, and incubate at 37℃ for 1 hour.
After the reaction was completed, the amount of reduced NAD produced by the reaction was measured as absorbance at a wavelength of 340 nm. As a result, as shown in FIG. 5, a good ATP calibration curve was obtained. Based on the results, calculated from the milli-extinction coefficient of reduced NAD, the reduced NAD has a 5 molar ratio to ATP.
It was produced by NAD. Example 2 [Creatine kinase activity measurement] 0.5M imidazole-HCl buffer (PH7.0) 0.1ml 40mM NAD 0.05ml 40mM CoASH 0.05ml 50mM MgCl ₂ 0.03ml 10mM linoleic acid (2% TritonX-100 (dissolved in)
0.1ml 1mM・FAD 0.01ml Acyl-CoA・Synthase activity containing solution (60U/
ml) 0.02ml Solution containing acyl-CoA/oxidase activity (150U/ml) 0.1ml Solution containing complex active enzyme (enoyl-CoA/hydratase activity 400U/ml, 3-hydroxyacyl-
CoA/dehydrogenase activity 200U/ml, 3-ketoacyl-CoA/thiolase activity 163U/ml)
0.1ml catalase 0.02ml 0.5M β-mercaptoethanol 0.01ml 10mM ADP 0.1ml 0.2M creatine phosphate 0.2ml Purified water 0.11ml total 1.0ml Add creatine kinase (manufactured by Boehringer; indicated) to 1.0ml of the reaction solution having the above composition. activity
Add 5μ of a 1μg/ml solution of 380U/mg) and
The reaction was carried out at ℃. After the reaction, the amount of reduced NAD produced by the reaction was measured over time using absorbance at a wavelength of 340 nm. The results are shown in FIG. Also, from this result 1
The increase in absorbance per minute is 0.034, and the creatine kinase activity is calculated from this value.
It was 222.0U/mg. The calculation formula is as follows. Activity (U/mg) = (increase value per minute)
× 1/6.1 A reaction solution for ADP quantification is obtained. In addition, by using creatine phosphate as a test solution in place of the above-mentioned creatine kinase test solution and using creatine kinase in place of creatine phosphate in the reaction solution, a reaction solution for quantifying creatine phosphate can be easily obtained. . Example 3 [Measurement of pyruvate kinase activity] To 1.0 ml of a reaction solution having the following composition, 5 to 20 μ of a solution of 0.1 U/ml of Sigma pyruvate kinase (type) was added to start the reaction, and the reaction was carried out at 340 nm at 37°C.
The increase in absorbance was measured over time. FIG. 7 shows the relationship between the increase in absorbance per minute from 3 minutes to 4 minutes and the amount of pyruvate kinase. As is clear from FIG. 7, a good linear relationship was obtained between the amount of enzyme and the increase in absorbance. 0.2M triethanolamine buffer (PH7.5)
0.15ml 10mM Phosphoenolpyruvate 0.05ml 25mM MgCl ₂ 0.1ml 100mM KCl 0.1ml 50mM ADP 0.1ml 50mM CoASH 0.05ml 10mM Linoleic acid (dissolved in 2% TritonX-100)
0.1ml 40mM NAD 0.05ml 1mM FAD 0.01ml Acyl-CoA/synthetase activity containing solution (60U/
ml) 0.02ml Solution containing acyl-CoA/oxidase activity (150U/ml) 0.1ml Solution containing complex active enzyme (enoyl-CoA/hydratase activity 400U/ml, 3-hydroxyacyl
CoA/dehydrogenase activity 200U/ml, 3-ketoacyl-CoA/thiolase activity 163U/ml)
0.1 ml Purified water 0.07 ml Total 1.0 ml Example 4 [Quantification of acetyl phosphate] To 1.0 ml of the reaction solution having the following composition, 20μ of 0.3 mM acetyl phosphate solutions at various concentrations were added, and the reaction was carried out at 37°C for 10 minutes. After the reaction, the amount of reduced NAD produced by the reaction was measured as absorbance at a wavelength of 340 nm. The results are shown in FIG. 0.2M triethanolamine buffer (PH7.4)
0.2ml 20mM ADP 0.1ml 40mM CoASH 0.05ml 40mM NAD 0.05ml 50mM MgCl ₂ 0.03ml 10mM Linoleic acid (dissolved in 2% TritonX-100)
0.10ml 1mM FAD 0.01ml Acyl-CoA/synthetase activity containing solution (60U/
ml) 0.02ml Solution containing acyl-CoA/oxidase activity (150U/ml) 0.1ml Solution containing complex active enzyme (enoyl-CoA/hydratase activity 400U/ml, 3-hydroxyacyl-
CoA/dehydrogenase activity 200U/ml, 3-ketoacyl-CoA/thiolase activity 163U/ml)
0.1ml Acetate Kinase (Sigma 230U/ml)
0.05ml Purified water 0.19mlTotal 1.0ml Example 5 [Quantification of ADP] To 1.0ml of the reaction solution having the following composition, add 20μ of ADP solutions of various concentrations from 0 to 0.5mM, and react at 37°C for 5 minutes. After that, the amount of reduced NAD produced by the reaction was measured as absorbance at a wavelength of 340 nm. As a result, good results were obtained as shown in Figure 9.
A calibration curve for ADP was obtained. 0.2M Pipes-NaOH buffer (PH7.2) 0.2ml 40mM NAD 0.05ml 40mM CoASH 0.05ml 50mM MgCl ₂ 0.03ml 10mM linoleic acid (dissolved in 2% TritonX-100)
0.1ml 1mM FAD 0.01ml 10mM phosphoenolpyruvate 0.1ml Acyl-CoA/synthetase activity containing solution (60U/
ml) 0.02ml Solution containing acyl-CoA/oxidase activity (150U/ml) 0.1ml Solution containing multiple active enzymes (enoyl-CoA/hydratase activity, 400U/ml, 3-hydroxyacyl-CoA/dehydrogenase activity 200U/ml, 3 −
Ketoacyl-CoA/thiolase activity 163U/ml)
0.1 ml pyruvate kinase (45 U/ml) 0.1 ml purified water 0.14 ml Total 1.0 ml [Example of production of multi-active enzyme] 100 ml of medium in a 500 ml Erlenmeyer flask (medium composition: peptone 1.5%, powdered yeast extract 0.5%,
KCl0.2%, _NaCl0.1 %, _K2HPO40.1 %,
MgSO ₄ 0.05%, oleic acid 0.75%, PH7.0; 15 bottles) and autoclaved at 120℃, then heated to 30℃.
Pseudomonas fragi B-0771 (FERM-P
No. 5701) was inoculated, and the rotary shaker was used for 20
Cultured for hours. The cultures were then merged,
Cultured bacterial cells were obtained by centrifugation at 5000 rpm for 20 minutes.
Furthermore, the obtained bacterial cells were added to 10ml containing 1.50mg of lysozyme.
In addition to 360 ml of M phosphate buffer (PH7.0), 340 ml of a soluble crude complex-active enzyme-containing solution (specific activity of 3-hydroxyacyl-CoA/dehydrogenase activity was 3.0 U/mg). I got it. After cooling 290 ml of the obtained crude composite active enzyme in an ice bath, 290 ml of acetone previously cooled to -20°C was added, the resulting precipitate was collected, and this was mixed with 1M KCl.
It was dissolved in a 10mM Tris-HCl buffer (PH7.5), and further centrifuged at 12,000 rpm for 10 minutes to remove insoluble materials. Aliquot 45 ml of the obtained supernatant,
Add 22.5ml of saturated ammonium sulfate solution (PH7.0) to this,
The resulting precipitate was removed by centrifugation at 12,000 rpm for 10 minutes. Next, 28 ml of saturated ammonium sulfate solution was further added to 57 ml of the obtained supernatant to cause precipitation. After collecting this precipitate, it was dissolved in 10 ml of 10 mM Tris-HCl buffer (PH 7.5) containing 1 M KCl and heated at 4°C against 2.0% of 10 mM Tris-HCl buffer (PH 7.5).
Dialyzed and desalted in a cellulose tube for 20 hours, this was then freeze-dried to obtain a composite active enzyme powder (3-
Hydroxyacyl-CoA dehydrogenase activity (specific activity was 18.2U/mg) 77mg
I got it. Furthermore, the obtained composite active enzyme powder (3-hydroxyacyl-CoA/dehydrogenase activity,
This specific activity was 18.2 U/mg. ) 77mg to 20ml
DEAE-Sepharose was dissolved in water and equilibrated with 10mM Tris-HCl buffer (PH7.5).
CL-6B (manufactured by Pharmacia) column (3 x 11
cm) for adsorption, and the column was washed with the same buffer solution. Then add 500ml of the above buffer and
Elution was performed using a linear concentration gradient method prepared with 500 ml of the same buffer containing 0.4 MKCl. At a flow rate of 52 ml/hour, 9 ml of each fraction was collected, the enzyme activity of each fraction was measured, and 90 ml of active fractions (Nos. 71 to 80) were obtained (3-hydroxyacyl-CoA/dehydrogenase activity). , its specific activity was 30.0 U/mg).
Furthermore, this active fraction was added to 10mM phosphate buffer (PH
7.5) After dialyzing against 2, it was charged and adsorbed onto a column (2 x 10 cm) filled with hydroxyapatite gel. Elution was performed by a linear concentration gradient method prepared with 300 ml of 10 mM phosphate buffer (PH 7.5) and 300 ml of 0.5 M phosphate buffer (PH 7.5). A 5 ml portion was collected at a flow rate of 33 ml/hour, the enzyme activity of each fraction was measured, and the active fraction (No. 46
~62) 85 ml was obtained (specific activity of enoyl acyl-CoA/hydratase activity was 210.3 U/mg, specific activity of 3-hydroxyacyl-CoA/dehydrogenase activity was 105 U/mg, 3-ketoacyl-CoA/
The specific thiolase activity was 85.6 U/mg).
This active fraction was then concentrated using an ultrafiltration membrane (XM-50 manufactured by Amicon), and then charged to a column (1.1 x 90 cm) packed with Toyo Pearl HW-60 (manufactured by Toyo Soda). (Solvent: 10mM Tris-HCl buffer PH7.5,
0.5MKCl). Aliquot 3.5 ml each and collect the active fraction (No. 23~
27) 17.5 ml was obtained and lyophilized to obtain a purified complex active enzyme (3-hydroxyacyl-
The specific CoA/dehydrogenase activity was 110 U/mg. Yield 20mg).

[Brief explanation of drawings]

Figure 1 is Pseudomonas fragi B-0771
(FERM-P No. 5701) shows a curve showing the optimum pH of the 3-hydroxyacyl-CoA dehydrogenase activity of the complex-active enzyme.
Figure 3 shows a curve showing the PH stability of dehydrogenase activity, Figure 3 shows a curve showing the thermostability of 3-hydroxyacyl-CoA dehydrogenase activity of the complex active enzyme, and Figure 4 shows reduced NAD by various fatty acids. Figure 5 shows the calibration curve for ATP, Figure 6 shows the increase in the amount of reduced NAD induced by creatine kinase over time, and Figure 7 shows the increase in the amount of reduced NAD induced by creatine kinase. Figure 8 shows a calibration curve for measuring pyruvate kinase activity by increasing the amount of reduced NAD; Figure 8 shows a calibration curve for acetyl phosphate;
The figure shows the calibration curve for ADP.

Claims (1)

[Scope of Claims] 1. In quantifying ATP in a test solution, the following reaction steps are performed: ATP is released from the kinase, ADP, and a phosphorus compound for the kinase substrate, or a test solution containing ATP is released. A reaction step in which fatty acids are reacted with CoASH and an enzyme having acyl-CoA synthetase activity to produce acyl-CoA.
A reaction step for converting dehydroacyl-CoA into hydrogen peroxide and dehydroacyl-CoA using an enzyme having CoA/oxidase activity, a reaction step for converting dehydroacyl-CoA to hydroxyacyl-CoA using water and an enzyme having enoyl-CoA/hydratase activity, hydroxyacyl -CoA, NAD and 3
- A reaction step for converting ketoacyl-CoA into reduced NAD and ketoacyl-CoA with an enzyme having hydroxyacyl-CoA/dehydrogenase activity, a reaction step for converting ketoacyl-CoA into acyl-CoA using an enzyme having CoASH and 3-ketoacyl-CoA/thiolase activities. In the reaction step including reagents and enzymes for the reaction, the acyl-CoA produced in the step is subjected to a cycling reaction as a substrate for the acyl-CoA in the step, and at least 2 Cycling the reaction for more than one cycle, then measuring at least one component of the amount of the consumed component or the amount of the component produced in a high molar ratio that can be detected depending on the number of cycles of the cycling reaction, and optionally step As an equivalent step, acyl-CoA was used instead of an enzyme with acyl-CoA oxidase activity in
dehydroacyl by an electron acceptor and an enzyme with acyl-CoA dehydrodenase activity
A quantitative method characterized by a reaction step including a step of converting CoA. 2, and the reaction step is enoyl-
A patent claim that is a reaction process based on the enzymatic activity of a multi-active enzyme having CoA/hydratase activity, 3-hydroxyacyl-CoA/dehydrogenase activity, and 3-ketoacyl-CoA/thiolase activity on the same protein. The quantitative method described in item 1. 3. The assay method according to claim 2, wherein the multi-active enzyme is an enzyme obtained from a multi-active enzyme producing bacterium belonging to Pseudomonas fragi. 4 A multi-active enzyme producing bacterium belonging to Pseudomonas fragi is Pseudomonas fragi B-0771.
The quantitative method according to claim 3, which is a bacterium. 5. Quantification according to claim 1, wherein the amount of reduced NAD produced is determined not by an absorption wavelength specific to NAD but by a wavelength in an absorption wavelength range that is an absorption wavelength specific to reduced NAD. Law. 6. The quantitative method according to claim 5, wherein the wavelength of the absorption wavelength range is around 320 nm to 360 nm. 7. The quantitative method according to claim 6, wherein the wavelength of the absorption wavelength range is around 340 nm. 8 Quantification of the amount of reduced NAD produced
2. The quantitative method according to claim 1, which is quantitative determination by color development of a hydrogen atom transport system chromogen capable of accepting hydrogen atoms of NAD. 9. The assay method according to claim 8, wherein the hydrogen atom transfer system capable of accepting hydrogen atoms of reduced NAD is a hydrogen atom transfer system containing diaphorase and a water-soluble tetrazolium salt. 10. The quantitative method according to claim 1, wherein the component in the test liquid is ATP. 11 Components in the test solution are liberated from the kinase, ADP, and the phosphorus compound for the kinase substrate.
The method for quantifying ATP according to claim 1. 12. The quantitative method according to claim 11, which is a measurement of one component of the following: measurement of kinase, measurement of ADP, and measurement of a phosphorus compound for a kinase substrate. 13 Kinase measurements include creatine kinase,
13. The quantitative method according to claim 12, which is a measurement of pyruvate kinase and acetate kinase. 14. The assay method according to claim 12, wherein the phosphorus compound for kinase substrate is creatine phosphate, phosphoenolpyruvate, or acetyl phosphate. 15 At least any two components of the following composition: kinase, ADP, and phosphorus compound for kinase substrate, - fatty acid, - CoASH, - NAD, - an enzyme having acyl-CoA synthetase activity, and - acyl-CoA oxidase activity. Claim 1 contains an enzyme having: - an enzyme having enoyl-CoA hydratase activity; - an enzyme having 3-hydroxyacyl-CoA dehydrogenase activity; - an enzyme having 3-ketoacyl-CoA thiolase activity. Described quantitative method.