Specific probe substrate of catechol-O-methyltransgerase and application thereof

A methyltransferase and probe substrate technology, applied in the field of medicine, can solve the problems of inconvenient quantitative detection, difficult separation and use, etc., and achieve the effects of good ultraviolet absorption characteristics, easy access, and sensitive detection.

Active Publication Date: 2013-07-10
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
View PDF0 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latter three cannot be used as in vivo probes because they are endogenous substances in the human body
In addition, the probe substrates of the above COMT enzymes include protocatechuic acid, which are catalyzed by mammalian COMT enzymes to generate two monomethylated products, and the two products are usually separated by liquid chromatography due to their similar structures and physical and chemical properties. Difficult to separate, which brings inconvenience to its quantitative detection
Therefore, there is an urgent need to find COMT enzyme probe substrates with single metabolites, sensitive detection, high safety, and clear metabolic pathways in vivo.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Specific probe substrate of catechol-O-methyltransgerase and application thereof
  • Specific probe substrate of catechol-O-methyltransgerase and application thereof
  • Specific probe substrate of catechol-O-methyltransgerase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Afracetin is used for the determination of COMT enzyme activity in 12 cases of individual human liver cytoplasm

[0030] Twelve commercialized human liver cytoplasmic samples from different individuals were purchased, and the enzyme activity of COMT in the human liver samples was determined using afracetin. The specific operation process is as follows:

[0031] (1) 40mM DTT and 5mM MgCl in 200 microliters of in vitro metabolic reaction system 2 , the concentration of human liver cytoplasm is 0.5mg / ml, the final concentration of afracetin is 100μM, pre-incubated at 37°C for 3 minutes;

[0032] (2) Add 10μl SAM (final concentration 4mM) to the reaction system to start the reaction;

[0033] (3) After 10 minutes, add 200 μl acetonitrile and shake vigorously to terminate the reaction;

[0034] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 20 minutes under the condition of 20,000×g, and then take the supernatant for HPLC-UV dete...

Embodiment 2

[0036] Example 2. Daphnetin is used to detect enzyme activity in human brain tissue homogenate S9

[0037] Daphnetin was used to detect the COMT catalytic activity in human brain tissue homogenate S9, and the specific steps were as follows:

[0038] (1) 40mM DTT and 5mM MgCl in 200 microliters of in vitro metabolic reaction system 2 , the concentration of S9 in human brain tissue is 0.5mg / ml, the final concentration of Daphnetin is 10μM, pre-incubated at 37°C for 10 minutes;

[0039] (2) Add 10μl SAM (final concentration 4mM) to the reaction system to start the reaction;

[0040] (3) After 10 minutes, add 200 μl acetonitrile and shake vigorously to terminate the reaction;

[0041] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under the condition of 20,000×g, and then take the supernatant for HPLC-UV detection and analysis;

[0042] Quantitatively detect the generation of glucuronic acid-conjugated products by ultraviolet absorpti...

Embodiment 3

[0043] Example 3.4-Methyldaphnetin is used for the determination of COMT enzyme activity in human erythrocytes

[0044] (1) Dilute the cells with red blood cell incubation solution, place them in a 6-well culture plate, 4ml per well, put them in a metal bath shaker, 80r / min, and incubate continuously at 40°C for 120 minutes;

[0045] (2) Add 4-methyl daphnetin to the culture plate with a final concentration of 50 μM;

[0046] (3) After 30 minutes, draw 200 μl of the incubation solution and place it in a -80°C ultra-low temperature refrigerator to stop the reaction;

[0047] (4) Add 200 μl of methanol to the sample to precipitate protein, and use a high-speed refrigerated centrifuge to centrifuge the above system at 20,000×g for 20 minutes, then take the supernatant for HPLC-UV detection and analysis.

[0048] HPLC-UV was used to detect fencetin and its metabolites respectively. The results showed that the sensitivity of HPLC-UV to detect fencetin and its metabolites could rea...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Login to view more

Abstract

The invention provides a specific probe substrate of catechol-O-methyltransgerase and application thereof. The specific probe substrate is a 7,8-dihydroxycoumarin compound and can be used for determination of activity of COMT enzyme in mammalian tissue and cells from different sources. The determination comprises the following concrete steps: selecting a coumarin compound having hydroxyl groups at positions 7 and 8 as a highly specific probe substrate; carrying out a COMT catalyzed reaction of the specific probe substrate in virtue of a COMT in-vitro incubation system; and determining the activity of COMT enzyme in each biological sample and each cell through quantitative detection of a product generation amount per unit time. The specific probe substrate can be used for quantitative evaluation of the activity of COMT enzyme in biological samples of different species and from different individual sources and quantitative determination of the activity of COMT enzyme in different-source-derived animal tissue cell culture fluids and prepared cell products, so assessment of capability of the important drug metablic enzyme COMT in disposition of drugs can be realized.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a specific probe substrate of a catechol-O-methyltransferase and an application thereof. Background technique [0002] Catechol-O-methyltransferase (COMT) is a methyltransferase widely distributed in mammals, and its main physiological function is responsible for the metabolism of endogenous neurotransmitters (such as epinephrine, norepinephrine hormones and dopamine, etc.) and exogenous catechol compounds. 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 amount of S-COMT is about 3-4 times that of MB-COMT. In the human brain, the COMT enzyme mainly exists in the form of MB-COMT, accounting for 7...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/16C12Q1/48
Inventor 杨凌夏杨柳葛广波孙晓宇宁静何桂元
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap