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Method for quantitative determination of catechol-o-methyl transferase activity and use thereof

A methyltransferase and quantitative determination technology is applied in the field of quantitative determination of catechol-O-methyltransferase activity, which can solve the problems of inconvenient quantitative detection, difficult separation by liquid chromatography, high cost and the like, and achieves good fluorescence emission. The effect of spectral characteristics, simple synthesis process and low detection cost

Inactive Publication Date: 2016-06-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-throughput detection of samples is difficult due to expensive chromatography or mass spectrometry methods and long sample analysis times
In addition, the probe substrates used in the above detection methods, including protocatechuic acid, generate two monomethylated products under the catalysis of mammalian COMT enzyme, and the two products are similar in structure and physical and chemical properties, and can be detected by liquid chromatography. It is usually difficult to separate, which brings inconvenience to its quantitative detection

Method used

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  • Method for quantitative determination of catechol-o-methyl transferase activity and use thereof
  • Method for quantitative determination of catechol-o-methyl transferase activity and use thereof
  • Method for quantitative determination of catechol-o-methyl transferase activity and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Synthesis of 4-Acetate-7-Hydroxy-8-Methoxycoumarin

[0039] The synthetic route of 4-acetic acid-7-hydroxyl-8-methoxycoumarin is as follows Figure 8 As shown, weigh 0.5g of 2-methoxyresorcinol and place it in a three-necked flask, add 0.6g of acetone-1,3-malonic acid, 1.5ml of perchloric acid, stir at room temperature for 20 minutes, and heat to 55 °C, react for 3 hours, TLC detects that the reaction is complete, the reaction solution is directly filtered, and the filter cake is collected, which is the crude product. The crude product was subjected to silica gel column chromatography to obtain 150 mg of 4-acetic acid-7-hydroxy-8-methoxycoumarin as a white solid. Structural formula such as figure 1 show its 1 H-NMR spectrum and 13 C-NMR spectrum as figure 2 with image 3 shown;

[0040] 1 HNMR (500MHz, d-DMSO) δ: 3.31 (s, 2H, CH2), 3.82 (s, 3H, OCH3), 6.22 (s, 1H, COCH=C), 6.89 (d, J=3Hz, 1H, ArH ), 7.34 (d, J=8Hz, 1H, ArH);

[0041] 13 CNMR (500MHz, d-DMSO...

Embodiment 2

[0043] Quantitative assessment of COMT activity in liver microsomes from different individual sources

[0044] (1) Select 12 cases of human liver microsomes (HLM) and dilute them to 10mg / ml to prepare the COMT metabolic reaction system, including Tris-HCl buffer (50mM) at pH 7.4, human liver microsomes (0.5mg / ml), Dithiothreitol 40mM, MgCl 2 50mM, the final concentration of 4-acetic acid-7,8-dihydroxycoumarin is 5μM, pre-incubated with shaking for 3 minutes at 37°C;

[0045] (2) Add 10 μl of SAM with a concentration of 4 mM to the reaction system to initiate the reaction;

[0046] (3) After 15 minutes, add 200 μl of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0047] (4) Use a high-speed refrigerated centrifuge at 4°C, 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant, perform fluorescence detection (Ex=320nm, Em=520nm), and substitute the obtained fluorescence intensity into the standard curve Afterwards, the metabolic ...

Embodiment 3

[0049] Determination of the lower limit of detection of COMT in vitro

[0050] The experiment was carried out on a microplate reader using a 96-well plate, 4-acetate-7,8-dihydroxycoumarin 5μM, S-adenosylmethionine 200μM, dithiothreitol 2mM, MgCl 2 5mM, COMT single enzyme 5ng / ml~100ng / ml, pH7.4 Tris-HCl buffer 50mM, total volume 100μL, incubated at 37°C for 1h and analyzed by microplate reader, the average value of each group was compared with that without COMT Compared with the control group, it was shown that 30 and 50ng of COMT were statistically significant (P<0.05), and the lower limit of detection of COMT was determined to be 30ng.

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Abstract

The invention discloses a method for quantitative determination of catechol-o-methyl transferase activity and a use thereof and belongs to the technical field of biological medicines. The method utilizes 4-acetoxy-7,8-dihydroxycoumarin as a COMT enzyme specific probe substrate. The substrate can be specifically catalyzed through the COMT enzyme to produce a 8-methoxy product, the 8-methoxy product can produce a fluorescence signal at wavelength of 520nm and through detection of fluorescence intensity change, COMT activity is detected quantificationally. The method can be used for quantitative evaluation of activity of COMT enzymes in biological samples with different sources and can also be used for external rapid screening of COMT inhibitors and assessment of an inhibition capability. The method can be used for quantitative assessment of real activity of COMT enzymes in various external biological samples, realizes inhibitor rapid screening and inhibition capability quantitative evaluation and can be used for evaluating catalytic activity of different types of COMTs and COMT mutants with different amino acid sequences and further evaluating a catechol drug metabolism capability. The method has the advantages of high selectivity, low price, acquisition easiness and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for quantitatively measuring the activity of catechol-O-methyltransferase and its application. Background technique [0002] Catechol-O-methyltransferase (COMT) is a methyltransferase widely distributed in mammals, and its main physiological function is to be responsible for the metabolism of endogenous neurotransmitters (such as epinephrine, norepinephrine hormones and dopamine, etc.) and exogenous catechol drugs (such as carbidopa, benserazide, apomorphine, dobutamine, fenoldopam, α-methyl-L-DOPA, isoproterenol and rimeterol, etc.). In the human body, COMT enzyme exists in two forms, one is soluble COMT (S-COMT) and the other is membrane-bound COMT (MB-COMT). COMT is distributed in various tissues of the human body, such as kidney, liver, lung, etc. In the human brain, it mainly exists in postsynaptic neurons. In most tissues of the human body, the a...

Claims

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Application Information

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IPC IPC(8): C12Q1/48
Inventor 杨凌夏杨柳葛广波王平钱星凯窦同意
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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