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Method and kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity

An active, transferase technology, applied in the field of chemistry, which can solve problems such as the inability to achieve high-throughput operations

Inactive Publication Date: 2010-12-15
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the technical problem to be solved in the present invention is to provide a method for measuring Nampt enzyme activity and a kit thereof, which is simple to operate and can realize High throughput operation

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  • Method and kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity
  • Method and kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity
  • Method and kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity

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Embodiment 1

[0071] The establishment and optimization of embodiment 1 enzyme activity detection method

[0072] The present inventor successfully expressed and purified Nampt recombinant protein from the prokaryotic E. coli system, and established a method for detecting Nampt enzyme activity. The reaction principle is as follows: figure 1 As shown, the Nampt enzyme converts the substrate nicotinamide (NAM) into the product nicotinamide mononucleotide (NMN); after two simple chemical reactions, NMN is converted into a fluorescent derivative that can be used at an excitation wavelength of 382nm, The maximum fluorescence signal was detected at emission wavelength 445nm ( figure 2 ).

[0073] The inventors investigated the influencing factors of each step of the detection reaction to obtain the optimal reaction conditions for each step. In order to investigate the influence of reaction time and reaction temperature in the second step of the detection reaction, 10 μl of 20% acetophenone and...

Embodiment 2

[0078] Embodiment 2 enzymatic characteristic analysis

[0079] In enzymatic analysis, the Michaelis constant K m It is a basic characteristic constant of an enzyme, which includes the properties of the enzyme binding and dissociation with the substrate. The Mie equation is: v=V max *[S] / (K m +[S]), where v is the reaction rate; K m is the Michaelis constant; V max is the maximum speed of the enzyme reaction; [S] is the substrate concentration. From the Mie equation, it can be seen that the Mie constant K m Equal to the substrate concentration at which the reaction rate reaches half of the maximum reaction rate.

[0080] The inventors investigated the relationship between the concentration of the substrate NAM and the fluorescence intensity of the final product. The substrate NAM was serially diluted and then reacted with 2 μg / ml enzyme at 37°C for 30 minutes, and the fluorescence intensity of the detection reaction product was measured. As shown in the figure, the subst...

Embodiment 3

[0087] The establishment and quality control of embodiment 3 high-throughput screening models

[0088] 3.1 High-throughput screening process:

[0089] The high-throughput screening process is as follows:

[0090] 1. The enzyme reaction system is 25 μl, and the concentration of various components is: 50mM Tris-HCl (pH7.5), 0.02% BSA, 12mM MgCl2, 2mM ATP, 0.4mM PRPP, 2mM DTT, 2μg / mlNampt, 0.2μM NAM, 20 [mu]M candidate compound and 2% DMSO. First add 0.5 μl of the DMSO solution of the candidate compound to the 96-well plate, then add 20 μl of the enzyme reaction mixture solution (enzyme reaction components other than the substrate), pre-incubate at 37°C for 5 minutes, then add 4.5 μl of the substrate NAM solution to Start the reaction, react at 37°C for 15 minutes and then heat at 95°C for 1 minute to terminate the enzyme reaction.

[0091] 2. After the enzyme reaction solution is cooled on ice, add 10 μl 20% acetophenone and 2M KOH in sequence, mix well on a vortex mixer, and...

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Abstract

The invention discloses a method and a kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity. The method comprises the following steps of: (1) establishing the reaction for converting the Nampt catalytic substrate of nicotinamide (NAM) into the product of nicotinamide mononucleotide (NMN); (2) terminating the reaction by a heating method; (3) adding hypnone and strong base for thorough reaction; (4) adding formic acid for thorough reaction; (5) detecting the fluorescence intensity at the location with the excitation wavelength of 326-426nm and the transmission wavelength of 410-520nm. The invention also provides a method and a system for high-flux sieving of a Nampt inhibitor or activator. The method comprises the following steps of: incubating the compound candidate and the reacting system for the Nampt (except the substrate of NAM) in a micropore plate; determining the Nampt activity in accordance with the steps. The method has the advantages of simple operation, moderate reaction condition and high-flux operation.

Description

technical field [0001] The invention belongs to the field of chemistry, in particular to a method for measuring Nampt enzyme activity and a kit thereof. The invention also provides a high-throughput screening method and screening system for Nampt enzyme inhibitors or activators. Background technique [0002] Nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin or pre-B cell clone enhancer factor PBEF) is an indispensable protein in mammalian life activities. Nampt-deficient homozygous mice (Nampt- / -) die early in embryonic development. It regulates the level of NAD coenzyme, an essential energy substance in mammalian cells, and has physiological functions such as promoting angiogenesis, anti-apoptosis, participating in the body's inflammatory response, promoting the differentiation and maturation of various cells, and potentially activating progenitor cells. It has a potential role in the prevention and treatment of diseases such as stroke. Tumor cells ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/48G01N21/64
Inventor 缪朝玉张偌瑜管云枫王培徐添颖徐学文
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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