A method for determining the enzyme activity of glutamine synthetase

Abstract

This invention relates to a technique for detecting glutamine synthetase (GS) activity in cells, specifically the ability of animal cells to synthesize glutamine (Gln) from GS protein expressed in their cells. Using 293T cells as the tool cell line, this invention obtains a crude enzyme mixture containing GS. The difference between the experimental group and the control group is the relative enzyme activity of GS. This method is simple to operate and is particularly suitable for detecting enzyme activity differences in GS mutants.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and cell biology. It relates to a mild method for extracting cellular proteins, suitable for subsequent determination of protease activity. Background Technology

[0002] Glutamine (Gln) synthase (GS) is the core of nitrogen metabolism in plants and animals, and is also the only enzyme in mammalian cells that can directly utilize NH4+. + Proteins that synthesize glutamine. Glutamine plays an important role in tumor metabolism, and is present in liver cancer, breast cancer, pancreatic cancer, and glioma. In glutamine-preferring cells, glutamine is highly dependent on GS enzyme activity.

[0003] Furthermore, an article in the *New England Journal of Medicine* reported that in two newborns with severe brain malformations leading to multiple organ failure and death, the R341C and R324C mutations in the GS enzyme resulted in near-total loss of glutaminase activity. Studies have shown that GS-KO mice also died on the third day after birth. This demonstrates the irreplaceable role of GS enzyme activity in organismal growth, and the study of GS enzyme activity deficiency caused by GS mutations is of great significance.

[0004] GS utilizes glutamic acid and NH4 + Glutamine is produced by consuming ATP. Simultaneously, GS also possesses glutaminase activity, catalyzing the reaction of glutamine and hydroxylamine to produce γ-glutamylhydroxyxamic acid. The γ-glutaminase reaction can be used to determine glutamine synthetase activity. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to easily and accurately determine the differences in enzyme activity among different mutants of GS.

[0006] This invention provides a simple and economical GS enzyme activity detection technology, comprising the following steps:

[0007] ① Transfect 2 μg of different GS (NCBI sequence number NM_001033044.4) mutant expression plasmids into 293T cells (transfection steps are as described on the ProteinTech website); ② Collect 293T cells into 1.5 ml EP tubes 36-48 h after transfection; ③ Follow the 1×10 6Add 150 μl of 50 mM imidazole (Maclean, I823673) at a ratio of 150 μl per cell, mix by pipetting, and incubate at -80°C for 30 minutes; ④ Remove and incubate at room temperature for 15 minutes to thaw, then centrifuge at 12000 rpm for 10 minutes; ⑤ Take 50 μl of supernatant into a new EP tube, add 0.5 times the volume of 2×SDS loading buffer (Coollab) as a Western blot sample; Take another 100 μl of supernatant into a new EP tube, add an equal volume of reaction solution 1 (50 mM imidazole, 20 mM Na2HPO4, 0.16 mM ADP, 50 mM Gln, 25 mM hydroxylamine, 2 mM MnCl2) to each centrifuge tube, and incubate at 37°C for 45 minutes. ⑥ Remove the sample and add 200 μl of reaction solution 2 (2.42% ferric chloride, 1.45% TCA, and 1.82% HCl); mix well. Centrifuge at 12000 rpm for 5 minutes; ⑦ Transfer the supernatant to a 96-well plate and measure the absorbance at 540 nm using a spectrophotometer. ⑧ Perform an immunoblotting assay on the protein sample obtained above, and calculate the final relative GS enzyme activity after obtaining the relative expression abundance of the GS mutant.

[0008] Key operational precautions for this invention include:

[0009] (1) The abundance of GS protein expressed by the GS mutant expression plasmid should not be too low, otherwise the difference in GS enzyme activity will not be obvious; if the difference in enzyme activity of the cell line is being tested, the number of cells required should be increased accordingly based on the expression of GS.

[0010] (2) A control group needs to be set up in the experiment. A control group needs to be set up for each sample. In the reaction solution 1 of the control group, Gln is replaced with ddH2O.

[0011] (3) To ensure data reliability, at least 3 repeat groups need to be set up;

[0012] (4) The reaction solution 1 and reaction solution 2 used are prepared fresh each time.

[0013] This invention uses cells as the material. The crude enzyme solution is treated with a reaction solution (50mM imidazole, 20mM Na2HPO4, 0.16mM ADP, 50mM Gln, 25mM hydroxylamine, 2mM MnCl2) and a stop solution (2.42% ferric chloride, 1.45% TCA, and 1.82% HCl). The absorbance of the sample at 540nm is then measured. This kit is simple to use and is particularly suitable for comparing the enzyme activity of different mutants of GS. Attached Figure Description

[0014] Figure 1 Changes in enzyme activity and corresponding protein expression in 293T cells overexpressing different amounts of GS;

[0015] Figure 2 Images of enzyme activity and corresponding immunoblot values ​​of GS wild-type and mutant R341C (GS arginine at position 341 is mutated to cysteine, which is a known defect in glutamine synthetase activity) overexpressed in 293T cells. Detailed Implementation

[0016] This invention discloses a method for detecting GS enzyme activity. Those skilled in the art can refer to this document and appropriately modify the technical parameters to achieve the desired result. These parameters include the selection of the cell strains, the protein concentration of the crude enzyme extract, and the incubation time at -80°C. Modifications to these parameters that have little impact on the results are considered to be included in this invention. The method and its application have been described in detail through examples, enabling those skilled in the art to easily apply the method described herein. Compared with existing technologies, this invention has the following advantages and effects: reagents are readily available, the method is simple and easy to implement, and it can rapidly and efficiently detect the enzyme activity of GS.

[0017] Example 1

[0018] Enzyme activity assay of GS-wild type (NCBI serial number NM_001033044.4)

[0019] Five 6 cm cell culture dishes were filled with 5 × 10⁵ cells. 5293T cells were transfected with 293T cells (Shanghai Cell Bank) GS expression plasmid (PcDNA3.0) using PEI transfection reagent (Proteintech, #PR40001) 24h later: the amount added to each culture dish was 1, 1.5, 2, 2.5 and 3 micrograms respectively (the plasmid construction method refers to [1] Liu, X., Chen, D., Chen, H., Wang, W., Liu, Y., Wang, Y., Duan, C., Ning, Z., Guo, X., Otkur, W., Liu, J., Qi, H., Liu, X., Lin, A., Xia, T., Liu, HX & Piao, HL (2021) YB1 regulates miR-205 / 200b-ZEB1axis by inhibiting microRNA maturation in hepatocellular carcinoma. 41, 576-595.). After 48 hours, the cells were washed with 1×PBS buffer (Maclean's) and collected into 1.5 ml EP tubes. The tubes were centrifuged at 1200 rpm for 5 minutes at room temperature. The supernatant was removed, and 150 μl of 50 mM imidazole buffer was added to each tube. The tubes were vortexed and placed in a -80°C freezer for 30 minutes. After 30 minutes, the tubes were removed and placed at room temperature for 15 minutes. The tubes were then centrifuged at 10000 rpm for 15 minutes. The supernatant obtained from the centrifugation was the crude enzyme extract. 40 μl of the extract was taken out as the immunoblotting sample and placed into EP tubes. An equal volume of 2×SDS loading buffer was added to each tube (5 EO tubes corresponding to 5 transfection groups). Take 50 μl of the remaining crude enzyme extract and place it in a new 1.5 ml EP tube. Add 50 μl of freshly prepared reaction solution 1 (50 mM Mimidazole, 20 mM Na2HPO4, 0.16 mM ADP, 50 mM Gln, 25 mM hydroxylamine, 2 mM MnCl2) (experimental group). Simultaneously, take 50 μl of the same solution and place it in another new 1.5 ml EP tube. Add 50 μl of freshly prepared reaction solution 1 (excluding glutamine, otherwise the same as reaction solution 1) as the control group. Replace Gln in reaction solution 1 with an equimolar amount of ddH2O. After heating in a 37°C water bath for 45 minutes, remove the tubes and add 200 μl of reaction solution 2 (2.42% ferric chloride, 1.45% TCA, and 1.82% HCl) to each tube of the experimental and control groups. At this point, a color reaction occurs in the experimental group, while no color change occurs in the control group. Vortex to mix and centrifuge at 10,000 rpm for 5 minutes. The resulting reddish-brown transparent supernatant (experimental group) is our test sample, and the resulting colorless transparent supernatant is the control group sample. The supernatants from the experimental and control groups are sequentially transferred to separate 96-well transparent plates, and the absorbance measured at 540 nm represents the relative enzyme activity of GS. Figure 1 The horizontal axis represents the transfection amount of GS, and the vertical axis represents the absorbance at a wavelength of 540 nm. Immunoblot detection of GS expression: GS size is 45 kDa, therefore a 10% SDS-PAGE gel was used for electrophoresis. After transfer, the PVDF-blot membrane was blocked with 5% skim milk (g / ml) for 30 minutes, washed with 1×TBST, and incubated overnight at 4°C with GS antibody (BD Biosciences, #610517, 1:1000 dilution). The next day, it was incubated with secondary antibody at room temperature for 1 hour (secondary antibody was mouse antibody, purchased from Sigma). A5795 ) and expose ( Figure 1 The expression band of GS was obtained, proving that the GS plasmid was successfully expressed.

[0020] The difference in absorbance between the experimental group and the control group is the relative enzyme activity of each GS.

[0021] The ratio of the relative enzyme activity of GS obtained for each sample to the gray value of the corresponding GS protein expression band is the absolute value of GS enzyme activity.

[0022] Example 2

[0023] Comparison of enzyme activity assays of GS-amino acid mutants

[0024] 5×10 mm slabs were laid in a 6-hole plate. 5 Two cells were transfected with different mutant plasmids of GS (PcDNA3.0) using PEI transfection reagent after 24 hours: WT (NCBI sequence number NM_001033044.4) (0.5, 2.0 μg) and R341C (0.5, 2.0 μg) (GS cells were transfected with arginine at position 341 to cysteine). Subsequent experimental procedures were the same as described in Example 1. The expression of different mutants of GS was obtained by immunoblotting. Figure 2 Below), the grayscale values ​​of GS were obtained by processing the images using the Fusion software built into the exposure instrument (GS wt: 0.5 μg (15), 2 μg (40); GS R31C: 0.5 μg (14), 2 μg (30)). The enzyme activity of different mutants of GS was obtained by comparing the grayscale values ​​with the grayscale values. Figure 2 superior).

Claims

1. A method for determining the activity of glutamine synthase, characterized in that: 1) Collect cells transfected with glutamine synthase (GS) for 36-48 hours in EP tubes, centrifuge, and discard the supernatant; then, according to a cell count of 1*102... 5 -1*10 6 Add 100-200 μL of 45-50 mM imidazole buffer, with a pH of 6.8-7.0; 2) After dispersing the cells, place them at -70 to -80°C for 30-40 minutes; remove them and place them at room temperature for 15-20 minutes, then run them at 10,000-120,000 rpm for 10-25 minutes; the supernatant is the crude enzyme solution. 3) Divide the remaining crude enzyme solution after taking out 30-50 μL of crude enzyme solution in the above steps into two equal parts: add one experimental group to an equal volume of reaction solution 1, and add the other control group to an equal volume of reaction solution 1 without glutamine Gln. Incubate in a 37°-38° water bath for 45-60 minutes. The composition of reaction solution 1 is 150 mM imidazole, 20 mM Na2HPO4, 0.16 mMADP, 50 mM Gln, 25 mM hydroxylamine, and 2 mM MnCl2. 4) Add two times the volume of reaction solution 2 to the sample containing reaction solution 1; mix well; centrifuge at 10000-12000 rpm for 5-10 minutes. The composition of reaction solution 2 is 2.42% ferric chloride by volume, 1.45% trichloroacetic acid by mass and volume, and 1.82% HCl by volume. 5) Take 100-200 μL of supernatant into each well plate and measure the absorbance at 540 nm. The difference between the experimental group and the control group is the relative enzyme activity of each GS.

2. The method according to claim 1, characterized in that: Take 30-50 μL of crude enzyme solution for protein quantification and subsequent immunoblotting. Use the software of the fusion chemiluminescence instrument to obtain the gray value of the protein expression band and obtain the relative amount of GS protein expression. The ratio of the relative enzyme activity of GS obtained for each sample to the gray value of the corresponding GS protein expression band is the absolute value of GS enzyme activity.

3. The method according to claim 1, characterized in that: The cells were 293T cells.

4. The method according to claim 1, characterized in that: The glutamine synthase is any one or more of the GS mutants.

Images