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Activity detection method for S-adenosylmethionine decarboxylase (SAMDC) and application thereof

A technology of adenosylmethionine decarboxylase and adenosylmethionine, which is applied in the field of biomedicine, can solve the problems of common laboratory difficulties, expensive medicines, and the inability to use AdoMetDC drug screening, etc., to achieve simple operation and high effect good effect

Active Publication Date: 2015-01-28
CHINA THREE GORGES UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is very sensitive and accurate, it is difficult to use in ordinary laboratories and cannot be used for high-throughput AdoMetDC drug screening because it involves isotope operations, requires special equipment and operations, and the price of drugs is relatively expensive.

Method used

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  • Activity detection method for S-adenosylmethionine decarboxylase (SAMDC) and application thereof
  • Activity detection method for S-adenosylmethionine decarboxylase (SAMDC) and application thereof
  • Activity detection method for S-adenosylmethionine decarboxylase (SAMDC) and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Use a 1.5mL EP test tube to mix R1715uL and R2235uL of the carbon dioxide determination reagent, then add 2uL of adenosylmethionine with a concentration of 500mM, and finally add AdoMetDC protease with a final concentration of 1uM to make the total volume of the mixed solution 1000ul. Start the reaction. The mixed solution was incubated in a water bath at 37 degrees Celsius for reaction. After 5 minutes, the solution was transferred to a transparent 96-well plate (200uL / well), and the light absorption of the system at a wavelength of 340nm was detected by a microplate reader and other instruments. The reactivity was detected by comparison with a control system without addition of AdoMetDC or adenosylmethionine.

[0028] figure 1 The middle represents the absorbance value without adding AdoMetDC, adding adenosylmethionine (SAM), AdoMetDC, without adding adenosylmethionine (SAM). It can be seen from the figure that when the reaction proceeds to generate carbon dioxide, ...

Embodiment 2

[0030] Use a 1.5mL EP test tube to mix R1715uL and R2235uL of the carbon dioxide determination reagent, and then add 2uL of adenosylmethionine (in this experiment, the designed concentration is 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10mM7 Index), and finally add AdoMetDC protease final concentration of 1uM, so that the total volume of the mixture is 1000ul, start the reaction. The mixed solution was incubated in a water bath at 37 degrees Celsius for reaction. After 30 minutes, the solution was transferred to a transparent 96-well plate (200uL / well), and the light absorption of the system at a wavelength of 340nm was detected by a microplate reader and other instruments. The reactivity was detected by comparison with a control system without addition of AdoMetDC or adenosylmethionine.

[0031]In this method, the concentration of adenosylmethionine is critical to the effect of the system. It is found through experiments that controlling the concentration of adenosylmethionine at 1 mM o...

Embodiment 3

[0034] Known AdoMetDC inhibitor MGBG utilizes the inhibitory effect figure that this method detects (such as image 3 shown). MGBG was dissolved in DMSO, so DMSO was used as a control. The lower the concentration, the weaker the ability to inhibit AdoMetDC, and therefore the lower the absorbance (more NADH consumption). The figure shows that, using this method, the inhibitory effect of the known inhibitor MGBG on the SAM reaction catalyzed by AdoMetDC can be effectively detected and shows a clear concentration dependence. Replace the previous one with the image below.

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Abstract

The invention relates to an activity detection method for S-adenosylmethionine decarboxylase (SAMDC). The method comprises the following steps: mixing a carbon dioxide determination reagent R1 mixed reagent and R2 mixed reagent in a 1.5mL EP test tube, then adding adenosylmethionine, finally adding SAMDC to obtain a mixed solution with total volume of 1000 microliters, and starting the reaction; performing the incubation reaction on the mixed solution for 1 to 30 minutes in a water bath with the temperature of 37 DEG C, then transferring the mixed solution to a 200 microliters / pore transparent 96-pore plate, detecting the light absorption of the system at the 340nm wavelength by utilizing instruments such as a microplate reader, and detecting the reaction activity of the mixture. The method is simple in operation and good in effect, and the activity of the SAMDC can be detected only by simple light absorption detection. The method is suitable for rapidly detecting the activity of AMDC and screening and designing an inhibitor.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to S-adenosylmethionine decarboxylase (S-adenosylmethionine decarboxylase; AdoMetDC), in particular to a method for detecting the activity of human source S-adenosylmethionine decarboxylase and drug screening Applications. Background technique [0002] Protein is one of the main components of organisms and the main substance to complete various life activities. Among various proteins, proteases are crucial to life activities, and almost all biochemical reactions in organisms are catalyzed by proteases. The activity of various proteases in organisms has a strict regulation mechanism. Once there is a problem with the regulation mechanism, the activity of proteases is too high, too low or completely inactivated, which will cause various diseases. Therefore, it has very important theoretical and practical significance to regulate the activity of protease by drugs to restore and maintain it at...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N21/33
CPCG01N21/33G01N33/573
Inventor 刘森廖陈曾王艳林占景琼
Owner CHINA THREE GORGES UNIV
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