KR101044634B1 - Stabilization Method of PGH2 and Activity Assay Method of mPGES-1 - Google Patents

Abstract

The present invention relates to a method for stabilizing prostaglandin H2 (PGH2) and a method for measuring activity of prostaglandin synthase (mPGES-1), and more specifically, a peroxy group of PGH2 is a hydroxyl group. to prevent the switch to; (PGF2 prostaglandin F _2), phosphine pomol rib Dick acid to prostaglandin H2 substrate decomposes prostaglandin F2; force to the stabilization method and the PGH2 substrate for prostaglandin H2 (PGH2), which was added to (phosphomolybdic acid PMA) The present invention relates to a method for measuring the activity of prostaglandin synthase (mPGES-1) using double enzyme activity measurement of two types of enzymes, formolibic acid (PMA) and prostaglandin dehydrogenase (15-PGDH) and mPGES-1. The method for measuring activity of mPGES-1 of the present invention is not only inconvenient to use conventional tin chloride (Tin chloride; SnCl ₂ ), but also in the UV-vis region without using an expensive antibody to quantify the reaction product PGE2. By absorbance, the activity of mPGES-1 can be easily measured. Accordingly, the present invention provides an effective and low-cost method for measuring the activity of mPGES-1, which is required for the mass screening of the inhibitory activity of mPGES-1, a target protein for the development of anti-inflammatory drugs.

Anti-inflammatory diseases, PGH2, PGE2, mPGES-1, phosphomolybdic acid

Description

Stabilization Method of PGH2 and Activity Assay Method of Staphization Method of Stabilization of Prostaglandin H2 (PPH2) and Prostaglandin Synthetase (MPPES-1)

The present invention relates to phosphomolybdic acid (PMA) and prostaglandin dehydrogenase, which are substances that stabilize prostaglandin H2 (prostaglandin H2; hereinafter referred to as "PGH2"), which are substrates of prostaglandin synthase (hereinafter referred to as "mPGES-1"). (Hereinafter referred to as "15-PGDH") to a method for measuring the activity of mPGES-1.

Prostanoids that regulate various physiological effects, such as inflammatory response and blood pressure control, are biosynthesized from arachidonic acid (FIG. 1). Prostaglandin H2 (PGH2) is synthesized by COX-1 / COX-2 from arachidonic acid, and various prostanoids such as prostaglandin E2 (hereinafter referred to as "PGE2"), TXA, and PGI are synthesized from these PGH2. PGH2, a type of prostanoid biosynthesized from such arachidonic acid, is converted into PGE2 by three kinds of biosynthetic enzymes of prostanoid E2, and these PGE2 are associated with an inflammatory response. These three PGE2 biosynthetic enzymes are cPGES-1, a water-soluble protein, and mPGES-1 and mPGES-2, a prostaglandin synthetase, a protein bound to two microsome membranes. Among these three types of PGE2 synthase, mPGES-1 is responsible for the synthesis of PGE2, which stimulates the inflammatory response by stimulation, and is considered as a target protein of inflammatory drugs (Murakami, M., Naraba, H., Tanioka, T., Semmyo, N., Nakatami, Y., Kojima, F. Iked, a T., Fueki, M., Ueno, A, Oh S. and Kudo, I. Regulation of prostaglandin E2 biosynthesis by inducible membrane associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. (2000) J. Biol. Chem . 275, 32783-32792).

As a method of measuring the activity of mPGES-1 used to search for inhibitors of mPGES-1, a method of measuring the amount of PGE2 generated after separation of a reaction product by HPLC has been developed. Is not suitable for use in the search for large quantities of active inhibitors of mPGES-1 (Per-Johan Jakobsson, Staffan Thore′N, Ralf Morgenstern, Bengt Samuelsson. Identification of human prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. (1999) Vol. 96, pp. 7220-7225).

In addition, the method of using the antibody against PGE2 is commercially available as a method for mass-detecting the activity of mPGES-1 (Journal of Biomolecule Screening, 2005, vol 10, page 599-605, Assay Design Kit , USA). The method has the advantage of using an assay design kit (USA) that can measure the activity of mPGES-1 by identifying a small amount of PGE2 generated using an antibody of PGE2, but requires a high cost. In addition, PGH2, which is used as a substrate of mPGES-1, reacts with tin chloride (SnCl ₂ ) within 30 seconds since its unstable peroxy group is easily decomposed naturally due to its low stability. Because it must terminate, it is limited to searching activities in large quantities. Therefore, this conventional method has limited the mass screening of inhibitors of mPGES-1.

Accordingly, there has been a demand for development of a method for measuring activity that is suitable for mass screening by increasing the stability of PGH2, which is a substrate of mPGES-1, as well as a method of measuring mPGES-1 activity at a lower cost.

Thus, the present inventors are trying to find a method for measuring mPGES-1 activity by increasing the stability of the substrate PGH2, PMA2 stabilizes the peroxy group of PGH2 PGH2 even during the reaction time of 30 minutes or more, which is the measurement time of the activity of mPGES-1 Sufficient conditions were established for the mass retrieval of mPGES-1 activity by confirming its stability to prevent degradation. In addition, 15-keto-prostaglandin E2 (hereinafter referred to as "15-keto-PGE2") and 1,4- using PGE2 and nicotin amide adenine dinucleotide (hereinafter referred to as "NAD ⁺ ") as substrates. 15-PGDH, an enzyme that synthesizes 1,4-dihydronicotin amide adenine dinucleotide (hereinafter referred to as "NADH"), was synthesized using mPGES-1 to measure the fluorescence of NADH. The present invention has been completed by developing a method to more easily measure the activity of mPGES-1.

It is an object of the present invention to provide a low cost activity measurement method that can be used for mass screening of inhibitors of mPGES-1. To this end, the present invention develops a method for measuring the progress of enzyme reaction by increasing the stability of PGH2 and measuring the amount of NADH produced by simultaneously using 15-PGDH and mPGES-1, thereby reducing the inhibitor of mPGES-1 at low cost. To provide a way to search.

In order to achieve the foregoing object, the present invention PGH2; peroxy groups are decomposed into hydroxyl prostaglandin F2 of (prostaglandin H2 PGH2) (prostaglandin F 2; PGF2) phosphine pomol rib diksan to prevent the transition to (phosphomolybdic acid; PMA) It provides a method of stabilizing PGH2 using.

In addition, the present invention provides a method for measuring activity of mPGES-1 using dual enzyme activity measurement of PMA and prostaglandin dehydrogenase (15-PGDH) and prostaglandin synthase (mPGES-1) on two PGH2 substrates. .

Hereinafter, the present invention will be described in detail.

The present invention provides a method for screening the activity of mPGES-1, which can effectively measure the activity of mPGES-1, a prostaglandin E2 (PGE2) synthase, a target protein of an anti-inflammatory drug. As described above, prostaglandin H2 (PGH2), which is a substrate of mPGES-1, is naturally degraded to a hydroxyl group and converted to PGF2 by peroxy group instability. Therefore, when PGH2 is measured when mPGES-1 activity is measured, There is a disadvantage in that the reaction must be terminated using tin chloride (SnCl ₂ ) within 30 seconds before decomposition. In addition, PGE2, which is a reaction product, has a very low absorbance in the fluorescence or UV-vis region, which cannot be measured by spectroscopic method. Thus, the present invention provides a method of sufficiently stabilizing the peroxy group of the substrate PGH2 using the phosphomolybdic acid (PMA) during the activity measurement time. In addition, 15-keto-PGE2 and 15-PGDH and mPGES-1, enzymes that produce 15-keto-PGE2 and NADH (1,4-dihydronicotinamide adenine dinucleotide) based on PGE2 and NAD ⁺ (nicotinamide adenine dinucleotide) A two-enzyme assay system used is provided to provide a method for easily measuring the fluorescence of one of the products, NADH, by fluorescence spectroscopy.

The present invention provides a method of increasing the stability of the substrate PGH2 during the activity measurement period using PMA as a substance to stabilize the peroxy group of PGH2 (prostaglandin H2; PGH2). Specifically, the present invention provides a method for stabilizing PGH2 using phosphomolybdic acid (PMA) to prevent the peroxy group of PGH2 from being decomposed to hydroxyl groups and converted to prostaglandin F ₂ (PGF2). In the stabilization method of the PGH2, the concentration ratio of the PGH2 and PMA is preferably 1: 1 to 100, and most preferably 6: 100.

In addition, the present invention provides a method for measuring activity of mPGES-1 using dual enzyme activity measurement of PMA and prostaglandin dehydrogenase (15-PGDH) and prostaglandin synthase (mPGES-1) on two PGH2 substrates. . Specifically, the enzyme catalyzing the reaction of synthesizing 15-keto-PGE2 and NADH using PGE2 and NAD ⁺ synthesized by mPGES-1 as a substrate by increasing the stability of the peroxy group of PGH2 using PMA as described above. Phosphorus 15-PGDH is used simultaneously with mPGES-1 to provide a method for measuring the fluorescence of the reaction product NADH.

The present inventors confirmed the property of PMA stabilizing the peroxy group of PGH2 and using this to maintain stability so that PGH2 does not decompose during the reaction time of mPGES-1 for 30 minutes or more. Sufficient conditions were established for searching. Specifically, the peroxy group of PGH2, which is a substrate of 6 μM mPGES-1, is increased in stability by 0.1 mM PMA and does not decompose into hydroxyl groups for at least 30 minutes at room temperature. Therefore, the reaction time of 30 minutes or more can be secured, and sufficient time required to search a large amount of compounds can be ensured.

In addition, after mass-expressing and purifying 15-PGDH from human 15-PDGH gene in Escherichia coli, it was used with mPGES-1 to establish a coupled-enzyme assay system. Through this, PGE2, a reaction product of mPGES-1, was converted into 15-keto-PGE2 by 15-PGDH, and NAD + was converted into NADH in this reaction. By measuring the fluorescence of NADH, the product of this reaction, the degree of generation of PGE2 can be confirmed indirectly.

As a result, the stability of PGH2 used as a substrate was increased, and it was confirmed that the reaction could be effectively performed even for a reaction time of 30 minutes or more. The degree of PGE2 production was indirectly generated through the fluorescence of NADH obtained by using two kinds of enzymes. By establishing a method of confirming that it was confirmed that the method developed in the present invention can be used to search for inhibitors of mPGES-1.

In the activity measurement method of mPGES-1 of the present invention, the activity measurement method is preferably to measure the fluorescence of NADH (1,4-dihydronicotinamide adenine dinucleotide) or NADPH by adding NAD ⁺ or NADP as a substrate in addition to the PGH2 substrate Iii) stabilizing the PGH2 substrate by reacting PMA with a PGH2 substrate; Ii) reacting the stabilized PGH2 substrate with an mPGES-1 enzyme to produce PGE2; Iii) reacting the produced PGE with 15-PGDH enzyme and NAD ⁺ substrate to produce 15-keto-PGE2 and NADH; And iii) measuring the fluorescence of the generated NADH (see FIG. 2 ). In this case, the fluorescence of the NADH is most preferably measured using an excitation wavelength of 340 nm and an emission wavelength of 468 nm.

Hereinafter, the present invention will be described in detail in the above order. Specifically, the method for measuring activity of mPGES-1 of the present invention was conceived in the following order.

E. coli is used to mass express and purify proteins from human mPGES-1 and 15-PGDH genes. The reaction conditions using the purified mPGES-1 and 15-PGDH proteins, the substrates of these proteins, PGH2 and NAD ⁺ , and a method of measuring the reaction product NADH are established.

Hereinafter, the activity measurement method of mPGES-1 of the present invention is composed of the following steps.

Step 1) Mass expression and purification of human mPGES-1 and 15-PGDH using E. coli;

mPGES-1 and 15-PGDH are preferably expressed in E. coli , but are not necessarily limited thereto. In addition, each histidine tag and glutathione tag of mPGES-1 and 15-PGDH can be used to measure the amount of protein expressed, respectively, a nickel-nitrilotriacetic acid (Ni-NTA) column. And glutathione-S-transferase (glutathione-S-transferase) column is preferably used to purify the expressed protein, but is not necessarily limited thereto.

The present inventors transformed E. coli Rosetta (DE3) with the expression vector of pPGES-1, mPGES-1, by Kim Woo-il et al., And expressed and purified human mPGES-1 in large quantities. The results of the expression and purification steps were analyzed using SDS-PAGE (see FIG. 3 ). In addition, Escherichia coli BL21 (DE3) pLysS was transformed with the expression vector of 15-PGDH which is pGEX-2T by the Percival method, and the human 15-PGDH was expressed in large quantities and purified therefrom. The results of the expression and purification steps were analyzed using SDS-PAGE (see FIG. 4 ).

Step 2) stabilizing PGH2 using PMA;

In order to suppress the natural degradation of PGH2, 100 μM of PMA was used to stably present PGH2 during the enzyme activity measurement time. Specifically, the stabilization of PGH2 is preferably a mixture of 6μM PGH2 and 100μM PMA in advance, but is not necessarily limited to this concentration.

The inventors stabilized the PGH2, which is an unstable substrate during the enzyme activity reaction using mPGES-1 and 15-PGDH purified in step 1, by treating PMA (see FIG. 5 ).

Step 3) optimizing activity measurement using mPGES-1 and 15-PGDH;

PG2 was indirectly measured by measuring the fluorescence of the reaction product NADH at excitation wavelength 340 nm and emission wavelength 468 nm using mPGES-1 using PGH2 as a substrate and 15-PGDH using PGE2 as a substrate. Check the generation of. Specifically, the activity measurement method using mPGES-1 and 15-PGDH is preferably reacted in the reaction solution containing mPGES-1 4 ㎍ 15-PGDH 3 ㎍ 2 mM NAD ⁺ , but is not necessarily limited to this concentration no. The amount of NADH generated is preferably, but not necessarily limited to, measuring the fluorescence of NADH at an excitation wavelength of 340 nm and an emission wavelength of 468 nm. That is, it is possible to measure the absorbance of NADH instead of fluorescence and also NADP instead of NAD ⁺ Etc. are all available in the present invention. As such, the degree of generation of PGE2 may be indirectly confirmed through fluorescence of NADH obtained by using two kinds of enzymes, and the mass retrieval of active inhibitors of mPGES-1 may be easily performed using the above method. .

In addition, the present inventors verified the activity measurement method of mPGES-1 of the present invention using MK-886 known to have inhibitory activity against mPGES-1 (see FIG. 6 ).

As described above, the method of measuring activity of mPGES-1 of the present invention is not only inconvenient to use conventional tin chloride (Tin chloride; SnCl ₂ ) but also UV without using an expensive antibody to quantify the reaction product PGE2. The activity of mPGES-1 can be easily measured by absorbance in the -vis region. Accordingly, the present invention provides an effective and low-cost method for measuring the activity of mPGES-1, which is required for the mass screening of the inhibitory activity of mPGES-1, a target protein for the development of anti-inflammatory drugs. In addition, the present invention provides an effective and low-cost method for measuring the activity of mPGES-1 required for the mass screening of the inhibitory activity of mPGES-1, a target protein for the development of anti-inflammatory drugs.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Expression and Purification of Human mPGES-1

Expression and purification of human mPGES-1 is described by Woo-Il Kim, et al., Expression and purification of human mPGES-1 in E. coli and identification of inhibitory compounds from a drug-library, BMB reports 2008; 41 (11): 808-813). Specifically, pPGES-1 (BMB reports 2008; 41 (11): 808-813), which is an expression vector of mutated mPGES-1, can be overexpressed in Escherichia coli developed by the present inventors.E. Coli Rosetta (DE3) (Novagen, USA), transformed E. coli was cultured in LB-agar plate containing 30 ㎍ / ml of kanamycine and 34 ㎍ / ml of chloramphenicol to express mPGES-1. Separation (5000x g, 20 min) was isolated and E. coli was disrupted using an ultrasonic sonicator (sonicator) and the cell membrane was separated by ultrafast centrifugation (100,000xg, 1 hr). The cell membrane was then washed with 4% Triton X-100 solution (15 mM Tris-HCl, pH 7.4, 0.5 mM EDTA (ethylenediaminetetraacetic acid), 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM glutathione, 4% Triton-). X 100, 150 mM NaCl, 10% glycerol) and passed through a nickel-nitrilotriacetic acid (Ni-NTA) (QIAGEN, Germany) column (5 mL). MPGES-1 bound to Ni-NTA column was separated by elution solution (15 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 0.2% Triton-X 100, 200 mM imidazole) It was. In order to purify the mPGES-1 with high purity, the mPGES-1 fraction separated using the Ni-NTA column was passed through a Q-sepharose column (GE healthcare, USA), and then the Q-Sepharose MPGES-1 bound to the column was separated into an elution solution (15 mM Tris-HCl, pH 7.4, 10% glycerol, 0.2% Triton-X 100) consisting of 0.1-0.7 M NaCl linear gradient. .

Results of the expression and purification steps of the mPGES-1 in E. coli were analyzed using SDS-PAGE ( FIG. 3 ). Lane M in Figure 3 is the molecular weight markers (molecular weight markers); Lane 1 is a lysate prior to mPGES-1 expression; Lane 2 is a lysate after expressing mPGES-1; Lane 3 is a cell membrane fraction of E. coli expressing mPGES-1; Lane 4 is Solubilized mPGES-1; Lane 5 is mPGES-1 purified using a Ni-NTA column; Lane 6 shows mPGES-1 purified using Q-sepharose column. As a result, it was confirmed that mPGES-1 was separated by more than 90% purity ( Fig. 3 , lane 6 ).

Example 2 Expression and Purification of Human 15-PGDH

Expression and purification of human 15-PGDH was based on the Percival method (M. David Percival, Continuous spectrophotometric assay amenable to 96-well plate format for prostaglandin E synthase activity, Analytical Biochemistry 313 (2003) 307-310). Specifically, pGEX-2T (GE Healthcase, USA), an expression vector of 15-PGDH, was transformed into Escherichia coli BL21 (DE3) pLysS (Novagen, USA) to be expressed in Escherichia coli, and then transformed Escherichia coli into ampicillin. (ampicillin) was cultured in 2X YT medium to which 50 ㎍ / mL was added to express 15-PGDH, and then E. coli was separated by centrifugation (5000x g, 15 min) and crushed by E. coli using an ultrasonic grinder. The solution was separated using centrifugation (10,000 × g, 20 min). The extract was passed through a GST-agarose (Peptron, Korea) column (5 mL). 15-PGDH bound to GST-agarose column was separated by elution solution (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione (GSH), 1 mM EDTA, and 0.1 mM dithiothreitol).

The results of the expression and purification steps of E. coli of 15-PGDH were analyzed using SDS-PAGE ( FIG. 4 ). In FIG. 4 , lane M is a molecular weight marker; Lane 1 is a lysate of E. coli expressing 15-PGDH; Lane 2 is supernatant after centrifugation; Lane 3 pellets after centrifugation; lane 4 is proteins that do not adhere to glutathione column resin; Lanes 5 are proteins washed out of glutathione column resin; Lane 6 is 15-PGDH purified using a glutathione column. As a result, it was confirmed that 15-PGDH was separated with a purity of 85% ( FIG. 4 , lane 6 ).

Example 3 Reaction of mPGES-1 with 15-PGDH

The enzyme activity reaction using mPGES-1 and 15-PGDH purified as in Examples 1 and 2 was carried out as follows. Specifically, 4 μg of mPGES-1 and 3 μg of 15-PGDH purified at room temperature were prepared in 185 μl of reaction solution [50 mM Tris-HCl (pH 7.5), 2 mM NAD ⁺ , 2 mM GSH and 0.1 mM DTT]. It was then added to a 96-well black nonbinding plate (Cayman chemical company, USA). The reaction starts with the addition of 15 μl of substrate solution (6 μM PGH2, 100 μM PMA) to the enzyme solution and after 30 minutes the amount of NADH generated is fluorescence with excitation wavelength 340 nm and emission wavelength 468 nm. Was measured using a fluorescent plate reader.

Example 4 Stabilization of PGH2 by Phosphomolybdic Acid (PMA)

PGH2, which is an unstable substrate when reacting with enzyme activity using mPGES-1 and 15-PGDH purified as in Examples 1 and 2, was stabilized during the reaction time by treatment with PMA. To determine the stabilizing effect of PGH2 by PMA, 3 μg of purified 15-PGDH at room temperature was added to 185 μl of reaction solution [50 mM Tris-HCl (pH 7.5), 2 mM NAD ⁺ , 2 mM GSH and 0.1 mM DTT. ] And added to a 96-well black nonbinding plate (Cayman chemical company, USA). Initiate the reaction by adding 15 μl of substrate solution (6 μM PGH2, 100 μM PMA) to the enzyme solution and measure the amount of NADH generated by fluorescence at 340 nm of emission wavelength and 468 nm of emission wavelength. Measurement was made using a reader ( FIG. 5 ). As a result, in the absence of mPGES-1 in Figure 5, the production of NADH was not measured until 40 minutes ( Fig. 5, open circle ), which indicates that phosphomolybdic acid stabilizes PGH2 and is converted to PGF2 until 40 minutes. Inhibition shows that PGF2 inhibits NADH production from oxidation by 15-PGDH. On the other hand, when 4 μg of mPGES-1 purified at room temperature and 3 μg of 15-PGDH were reacted with PGH2, which is a substrate, in the presence of 100 μM of PMA, the PGE2 produced by mPGES-1 was oxidized by 15-PDGH. It was found that fluorescence was generated by generating NADH ( FIG. 5, filled circle ). This fluorescence change can be seen that the generation and oxidation of PGE2 is completed within a relatively fast time within 5 minutes, and then fluorescence is generated stably. It is possible to stabilize PGH2 for about 40 minutes by PMA, thus confirming that mPGES-1 activity is suitable for mass screening of compounds using multi-well plates.

Example 5 Validation of a Novel mPGES-1 Activity Assay Using MK-886

In order to verify the activity measurement method of mPGES-1 developed in the present invention, MK-886 (Mancini, JA, Blood, K., Guay, J., Gordon, R., known to have inhibitory activity against mPGES-1). , Claveau, D., Chan, CC, and Riendeau, D. (2001) J. Biol. Chem. 276, 4469-4475).

Specifically, 4 μg of mPGES-1 and 3 μg of 15-PGDH purified at room temperature were prepared in 185 μl of reaction solution [50 mM Tris-HCl (pH 7.5), 2 mM NAD ⁺ , 2 mM GSH and 0.1 mM DTT]. After MK886 was added at a concentration of 0.1 μM ~ 100 μM and reacted for 30 minutes. The reaction was started by adding 15 μl of substrate solution (6 μM PGH2, 100 μM PMA) to the reaction solution to which MK-886 reacted. After 30 minutes, the amount of generated NADH was fluorescence with excitation wavelength of 340 nm and emission wavelength of 468 nm. Was measured using a fluorescent plate reader. As a result, the concentration of MK886 (IC ₅₀ ) at which 50% inhibition of mPGES-1 activity was measured was 2.8 μM, which is a method of measuring mPGES-1 activity using conventional PGE2 antibody (Journal of Biomolecule Screening, 2005, vol 10 3.2 μM, IC ₅₀ value of MK886 measured by JA Mancini, K. Blood, J. Guay, R. Gordon, D. Claveau, CC Chan, D. Riendeau, Cloning, expression, and up- regulation of inducible rat prostaglandin E synthase during lipopolysaccharideinduced pyresis and adjuvant-induced arthritis, J. Biol. Chem. 2001, vol. 276, pp4469-4475). These results show that the method of mPGES-1 established in the present invention is consistent with the results of the conventional methods of measuring activity of mPGES-1, and thus, the method of measuring mPGES-1 activity of the present invention is effective in identifying mPGES-1 inhibitors. It was confirmed that it can be effectively used to search ( Fig. 6 ).

1 is a schematic diagram of a biosynthetic pathway in which prostagladin, a type of prostanoid, is synthesized from arachidonic acid.

2 is a schematic diagram of a method for measuring mPGES-1 activity using mPGES-1 and 15-PGDH according to the present invention;

3 is a photograph analyzed using SDS-PAGE according to the expression and purification step of Escherichia coli mPGES-1,

4 is a photograph analyzed using SDS-PAGE according to the expression and purification steps of E. coli of 15-PGDH,

5 is a graph measuring the stabilizing effect of PGH2 by phosphomolybdic acid (PMA) by fluorescence of NADH (wherein (●) (filled circle) is a substrate PGH2, NAD as a substrate in the presence of phosphomolybdic acid) ^{When +} reacts with mPGES-1 and 15-PGDH, PGE2 produced is oxidized by 15-PGDH and NADH is produced, and (○) (open circle) is a substrate in the absence of mPGES-1. When reacted with PGH2, NAD ⁺ and 15-PGDH, phosphomolybdic acid stabilizes PGH2 and inhibits conversion to PGF2 for up to 40 minutes, indicating that PGF2 inhibits the formation of NADH by oxidation reaction by 15-PGDH. ),

6 is a graph of NADH fluorescence measured using the mPGES-1 activity measuring method of the present invention that MK-886, a compound having inhibitory activity of mPGES-1, inhibits mPGES-1 activity at various concentrations.

Claims (6)

A method for stabilizing prostaglandin H2 (PGH2) by adding phosphomolybdic acid (PMA) to a prostaglandin H2 (PGH2) substrate. The method of claim 1, wherein the concentration ratio of prostaglandin H2 (PGH2) and phosphomolybdic acid (PMA) is 1: 1 to 100, characterized in that stabilization method of prostaglandin H2 (PGH2). delete delete Iii) stabilizing the prostaglandin H2 (PGH2) substrate by reacting phosphomolybdic acid (PMA) with the prostaglandin H2 (PGH2) substrate; Ii) reacting the stabilized prostaglandin H2 (PGH2) substrate with prostaglandin synthase (mPGES-1) to produce prostaglandin E2 (PGE2); Iii) reacting the produced prostaglandin E2 (PGE2) with prostaglandin dehydrogenase (15-PGDH) enzyme and nicotinamide adenine dinucleotide (NAD ⁺ ) substrate to react with 15-keto-prostaglandin E2 (15-keto-PGE2) Producing 1,4-dihydronicotinamideadenine dinucleotide (NADH); And Iii) measuring the fluorescence of the produced 1,4-dihydronicotinamide adenine dinucleotide (NADH); measuring the activity of prostaglandin synthase (mPGES-1) comprising a. The method of claim 5, wherein the fluorescence of the 1,4-dihydronicotinamide adenine dinucleotide (NADH) is measured using an excitation wavelength of 340 nm and an emission wavelength of 468 nm. Method for measuring activity of prostaglandin synthase (mPGES-1).

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