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Phosphatidyl ethanolamine N-methyltransferase activity detecting method

A technology of phosphatidylethanolamine and methyltransferase, which is applied in the fields of biomedicine, clinical diagnosis and detection, and can solve the problems that the detection method of enzyme activity is not easy to popularize.

Inactive Publication Date: 2016-05-18
北京中科卓明生物医学研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Therefore, based on the defects of the above-mentioned prior art, in order to solve the problem that the enzyme activity detection method of existing phosphatidylethanolamine N-methyltransferase (PEMT, PhosphatidylethanolamineN-methyltransferase, EC2.1.1.17) is not easy to popularize, the purpose of the present invention It provides a rapid, specific, accurate and reliable detection method for PEMT enzyme activity

Method used

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  • Phosphatidyl ethanolamine N-methyltransferase activity detecting method
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  • Phosphatidyl ethanolamine N-methyltransferase activity detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Crude Extraction of Rat Liver Endoplasmic Reticulum

[0087] Weigh 20 g of rat liver tissue from which adipose tissue has been excised, add 200 mL of 0.25 M (containing 0.01 M, pH 8 phosphate buffer) sucrose solution, and homogenize 20 times with a glass homogenizer. Sigma3-30K centrifuge at 2600 Centrifuge at 2800 rpm for 10 minutes, filter through four layers of gauze, and discard the precipitate. The supernatant was centrifuged at 15,000rpm for 30 minutes, and the resulting precipitate was the part containing the endoplasmic reticulum, which was suspended in 0.25M (containing 0.01M, pH8.0 phosphate buffer) sucrose solution, and the endoplasmic reticulum could be stored at -80°C Save it.

Embodiment 2

[0088] Example 2: Determination of phosphatidylethanolamine N-methyltransferase enzyme activity in rat liver endoplasmic reticulum (pre-treatment)

[0089] The PEMT detection reagent of the present embodiment is three doses of reagents, comprising

[0090] Reagent 1

[0091] Tris-hydrochloric acid buffer (pH8.5) 30mmol / L

[0092] SAM0.1mmol / L

[0093] Reagent 2

[0094] Tris-hydrochloric acid buffer (pH8.5) 30mmol / L

[0095] PE0.2mmol / L

[0096] Reagent 3

[0097] Potassium phosphate buffer (pH8.0) 30mmol / L

[0098] S-adenosyl-L-homocysteine ​​hydrolase 10KU / L

[0099] The PEMT sample to be tested is the extracted endoplasmic reticulum of rat liver tissue. After crushing, add sodium cholate solution to the sample to make the final volume percentage concentration reach 0.5%. After mixing gently, place it on ice for 30 minutes to wait. use. The total protein dosage in the reaction system is 10ug, and the reaction system is 250ul. A total of 5 parallel samples were ma...

Embodiment 3

[0106] Embodiment 3: Determination of enzyme activity of phosphatidylethanolamine N-methyltransferase in rat liver endoplasmic reticulum (sample is processed in the early stage comparison)

[0107] The phosphatidylethanolamine N-methyltransferase detection reagent of this embodiment is three reagents, including

[0108] Reagent 1

[0109] Tris-hydrochloric acid buffer (pH8.5) 30mmol / L

[0110] SAM0.1mmol / L

[0111] Reagent 2

[0112] Tris-hydrochloric acid buffer (pH8.5) 30mmol / L

[0113] PE0.2mmol / L

[0114] Reagent 3

[0115] Potassium phosphate buffer (pH8.0) 30mmol / L

[0116] S-adenosyl-L-homocysteine ​​hydrolase 10KU / L

[0117] The sample of phosphatidylethanolamine N-methyltransferase is the extracted endoplasmic reticulum of rat liver tissue. %, after mixing gently, place it on ice for 30min; add the same amount of sample suspension to another part, mix gently and place it on ice for 30min, the total protein dosage in the reaction system is 10ug, and the rea...

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Abstract

The invention provides a phosphatidyl ethanolamine N-methyltransferase activity detecting method. The method comprises the steps that a sample containing phosphatidyl ethanolamine N-methyltransferase makes contact with an S-adenosine-L-methionine and methyl acceptor to form methylate of the S-adenosine-L-methionine and methyl acceptor. S-adenosine-L-homocysteine makes contact with S-adenosine-L-homocysteine hydrolase to generate homocysterine and adenosine, and a photometer is adopted to evaluate the generation rate of adenosine so as to detect enzyme activity of phosphatidyl ethanolamine N-methyltransferase in the sample. The method has the advantages of being convenient, fast, high in sensitivity and convenient to apply and popularize.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and at the same time belongs to the field of clinical diagnosis and detection. Specifically, the present invention provides a method for measuring phosphatidylethanolamine N-methyltransferase (PEMT, PhosphatidylethanolamineN-methyltransferase, EC2.1.1.17) enzyme activity. Background technique [0002] Choline is a strong organic base. It is a component of lecithin and also exists in sphingomyelin. important role in fetal development. Choline has an affinity for fat, which can make fat be transported from the liver through the blood in the form of phospholipids, thereby preventing the formation of fatty liver, effectively protecting and treating liver fibrosis caused by alcohol, and has the effect of anti-hepatic fat peroxidation, thereby reducing alcohol. The apoptosis of liver cells caused by choline can regulate the activity of liver cells, reduce the cytotoxicity of tumor necrosis factor...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/34
CPCC12Q1/48C12Q1/34G01N2333/91017
Inventor 王天泽菅晓勇
Owner 北京中科卓明生物医学研究所有限公司
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