Separation and identification method of cAMP synthesis reaction system components and application thereof

Abstract

The invention discloses a separation and identification method of cAMP synthesis reaction system components and an application thereof, and aims at solving a technical problem that the ACs identification accuracy is low. The method comprises the following steps: taking ATP, ADP, AMP, cAMP and Ado standard substances, sampling to enter a high-performance liquid chromatograph to separate, and detecting appearance time; taking to-be-detected sample to add in a reaction buffer solution, taking supernatant to sample into the liquid chromatograph; contrasting the standard substance appearance time to judge the generation of cAMP; taking the to-be-detected sample into the reaction buffer solution to obtain a reaction solution; preparing the standard substance solution by taking the cAMP standardsubstance, thereby manufacturing a standard curve; sampling UPLC by taking the reaction solution and the standard substance solution; contrasting the TCI peak area in 135.8m / z with the standard curve,and computing cAMP content. Through the method disclosed by the invention, the accuracy of detecting the ultra-low cAMP content is obviously improved, the influence on the cAMP detection by Ado is effectively removed, and a strict, reliable an convenient method is provided for identifying the ACs.

Description

technical field [0001] The invention relates to the technical field of biological enzyme detection, in particular to a method for separating and identifying components of a cAMP synthesis reaction system and its application. Background technique [0002] In eukaryotes, adenylate cyclase (Adenylate cyclase, ACs) is an important component of the adenylate cyclase signaling system, which is responsible for catalyzing the second messenger 3',5'-cyclization of adenosine mono Phosphate (Cyclic Adenosinemonophosphate, cAMP) production, which affects protein kinase A (Protein kinase A, PKA), cAMP-activated exchange protein (Exchange protein directly activated by cAMP, EPAC), CRE-bound transcription factor (cAMP response element -binding protein, CREB) and cyclic nucleotide-gated ion channels (Cyclic nucleotide-gated ion channels, CNGCs) and other activities, involved in various life activities. [0003] In mammals, ACs are a large family. However, in plants, the knowledge of adeny...

AI Technical Summary

Problems solved by technology

Although this method can directly observe and quantify the production of cAMP, the test cost is low, and the test is flexible, but there are still deficiencies in the identification of ACs

For example, for a long time, cAMP can only be detected alone, and the reaction substrates ATP (ADP and AMP) have not been combined for detection. In addition, the structures of AMP, Ado (Adenosine) and cAMP are similar, and the existing LC cannot be ruled out when detecting cAMP. Interference between them, those skilled in the art generally believe that AMP, Ado and cAMP cannot be separated [A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice, TheJournal of Biological Chemistry, 287(6), 2012. 4023-4032; An Arabidopsis Clathrin Assembly Protein with a Predicted Role in Plant Defense Can Functionas an Adenylate Cyclase, Biomolecules, 2018, doi:10.3390 / biom8020015; The Arabidopsis thaliana K + -uptake permease 7 contains functional cytosolicadenylate cyclase catalytic centre, FEBS Letters,589, 2015, 3848-3852]

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