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Method for determining kinetic parameters of lysophosphatidic acid acyltransferase

A technology of lysophosphatidic acid and acyl transfer, applied in acyltransferase, transferase, chemical instruments and methods, etc., can solve the problems of high substrate and human disadvantage, and achieve harm avoidance, high-sensitivity detection ability, and high-efficiency separation ability. Effect

Pending Publication Date: 2022-01-28
ZHONGBEI UNIV
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Problems solved by technology

[0002] The LPAAT gene family and its homologous genes belong to the superfamily of membrane-bound acyltransferases. LPAAT is a key intermediate in the synthesis of phospholipids and triacylglycerol, and plays an important role in the production of PA. Therefore, the determination of LPAAT enzyme activity in vitro can Accurately understand the physiological functions of these enzymes. In plants, LPAAT is closely connected with chloroplasts, endoplasmic reticulum membranes and mitochondrial membranes. In Arabidopsis plastids, lysophosphatidic acid transferase is a key regulator of embryonic synthesis. Many genes are in The production of different types of lipid intermediates in different organs or tissues has great differences in function and activity. Therefore, the parameter determination, substrate preference determination and activity determination of lysophosphatidic acid acyltransferase in vitro are very meaningful, but this At present, the determination of enzymes mostly relies on radioisotope technology. The substrate of this method is high, which is not good for the human body, and the traditional method can only detect the reaction parameters of the same enzyme in the same reaction.

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  • Method for determining kinetic parameters of lysophosphatidic acid acyltransferase
  • Method for determining kinetic parameters of lysophosphatidic acid acyltransferase

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Embodiment Construction

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0021] see Figure 1-2 , an embodiment provided by the present invention: the method for measuring the kinetic parameters of lysophosphatidic acid acyltransferase comprises the following steps: Step 1, leaf transient expression and vector construction; Step 2, Agrobacterium-mediated transient expression in Ben's leaves Agrobacterium strain; step 3, extraction of leaf cell microsomes; step 4, in vitro LPAAT reaction; step 5, LC-MS lipidomics determination;

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Abstract

The invention discloses a method for determining kinetic parameters of lysophosphatidic acid acyltransferase. The method comprises the following steps: 1 transient expression of leaves and vector construction; 2 transient expression of agrobacterium strains on Benyuan leaves through agrobacterium mediation; 3 extraction of leaf cell microsomes; 4 in-vitro LPAAT reaction, and 5 LC-MS (liquid chromatography-mass spectrometry) lipomics determination. In the first step, the p19 gene is a tomato bush dwarf virus silencing suppressor, an LC-MS non-radioactive detection technology is adopted for determining the kinetic parameters of lysophosphatidic acid acyltransferase, efficient separation capacity and high-sensitivity detection capacity are achieved, different lipid types can be quantified in single analysis, the method is simple and rapid, and avoids the harm of radioactive substrates to a human body; and meanwhile, high-throughput analysis on the target lipid compound is realized by combining a triple quadrupole technology, and the dynamic range of the target type can be quantified according to factors such as the acquisition speed of a mass spectrometer and the like.

Description

technical field [0001] The invention relates to the technical field of enzyme kinetic analysis, in particular to a method for measuring kinetic parameters of lysophosphatidic acid acyltransferase. Background technique [0002] The LPAAT gene family and its homologous genes belong to the superfamily of membrane-bound acyltransferases. LPAAT is a key intermediate in the synthesis of phospholipids and triacylglycerol, and plays an important role in the production of PA. Therefore, the determination of LPAAT enzyme activity in vitro can Accurately understand the physiological functions of these enzymes. In plants, LPAAT is closely connected with chloroplasts, endoplasmic reticulum membranes and mitochondrial membranes. In Arabidopsis plastids, lysophosphatidic acid transferase is a key regulator of embryonic synthesis. Many genes are in The production of different types of lipid intermediates in different organs or tissues has great differences in function and activity. Therefor...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/54C12N15/40C12Q1/48C12Q1/30G01N30/02G01N30/72
CPCC12N15/8205C12N9/1029C07K14/005C12Q1/48C12Q1/30C12Y203/01051G01N30/02G01N30/72C12N2770/38022G01N2333/91057
Inventor 侯天宇李会珍张志军胡楠邢云周雪荣
Owner ZHONGBEI UNIV
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