Enzyme activity determination method of acetyl coenzyme A carboxylase, kit and application

Abstract

The invention relates to the technical field of substance detection, and particularly discloses an enzyme activity determination method of acetyl-CoA carboxylase, a kit and application. The invention provides an enzyme activity determination method of acetyl coenzyme A carboxylase. The detection reaction process is as follows: # imgabs0 #. The detection method provided by the invention can realize rapid and accurate detection of ACC enzyme activity, can perform efficient ACC determination in research and application, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of substance detection, and in particular to a method for determining the activity of acetyl-CoA carboxylase, a kit and application thereof. Background Art

[0002] Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to generate malonyl-CoA in the body and is a key enzyme in the synthesis of fatty acids and many secondary metabolites. The activity of ACC determines the synthesis rate of fatty acids and the oil content to a certain extent.

[0003] The current method for detecting ACC enzyme activity is that acetyl CoA, NaHCO3 and ATP are catalyzed by ACC to generate malonyl CoA, ADP and inorganic phosphorus. The phosphorus determination reagent and the product inorganic phosphorus generate a substance with a characteristic absorption peak at 660nm. The ACC enzyme activity is calculated by measuring the increase in inorganic phosphorus.

[0004] However, this method has some problems. (1) ATP, one of the substrates of the enzymatic reaction, will react with the phosphorus determination reagent, which will affect the accuracy of the experimental results. (2) There is a large amount of inorganic phosphorus in the animal and plant samples. If the background inorganic phosphorus exceeds the linear range of the measurement, the sample needs to be diluted to make the measured value within the linear range. If the enzyme activity itself is low, the dilution will cause the measurement result to be unstable or even negative, affecting the accurate measurement of ACC activity. (3) This method requires two-step reactions (enzymatic reaction and phosphorus determination reaction), which is time-consuming, requires the preparation of a standard curve, and requires multiple steps for measurement, which will also increase the complexity of the experiment. (4) The two-step enzymatic reaction cannot detect the dynamic changes of the reaction in real time. Therefore, it is necessary to further study the determination method of ACC enzyme activity. Summary of the invention

[0005] One of the purposes of the present invention is to provide a novel method for determining ACC activity with high accuracy.

[0006] The present invention provides a method for determining the enzyme activity of acetyl-CoA carboxylase, and the detection reaction process is as follows: .

[0007] The present invention provides a new method for determining the activity of acetyl-CoA carboxylase (ACC), which no longer reflects the activity of ACC through the content of inorganic phosphorus, but determines the activity of ACC through further reaction of ADP. The specific technical means of detecting the activity of ACC in the present invention are as follows: first, ACC carboxylates acetyl-CoA in the reaction system to generate malonyl-CoA, ADP and inorganic phosphorus; further, pyruvate kinase and lactate dehydrogenase are used to catalyze NADH to generate NAD + , and the activity of ACC was reflected by measuring NADH at a wavelength of 340 nm.

[0008] The method of the present invention is not affected by the inorganic phosphorus content in the sample to be tested, the experimental result is more accurate, and there is no need to prepare a standard curve. Only two tests of the sample to be tested and the blank sample are required to quickly obtain the test result, thereby improving the detection efficiency.

[0009] The method of the present invention comprises: mixing a sample to be tested with a reagent 1, a working solution and a reagent 2 to obtain a first reaction system, detecting the absorbance value at 340 nm within 30 seconds, and calculating it as an A1 determination; Then, the first reaction system was placed at 37±1℃ for 5-10min, and the absorbance was measured, which was calculated as A2 measurement; Determination, Determination = A1 determination - A2 determination; In addition, the sample to be tested in the first reaction system is replaced with distilled water to obtain a second reaction system, the volume of the second reaction system is the same as that of the first reaction system, and the A1 blank and A2 blank are obtained in the same manner as that of the A1 determination and the A2 determination; Calculate blank, Blank = A1 blank - A2 blank; pass Calculate the activity of acetyl-CoA carboxylase in the sample to be tested. = Determination - blank; The reagent 1 comprises: potassium salt, magnesium salt, NaHCO3, dithiothreitol, ATP and buffer solution; The second reagent includes: acetyl coenzyme A and water; The working solution includes reagent three, reagent four, reagent five, and reagent six; The reagent three includes pyruvate kinase and water; The reagent 4 includes lactate dehydrogenase and water; The reagent five includes phosphoenolpyruvic acid and water; The reagent six includes NADH and water.

[0010] In the reagent 1 of the method of the present invention, the concentration of potassium salt is 0-1.6 mg / mL, the concentration of magnesium salt is 0.4-0.9 mg / mL, the concentration of NaHCO3 is 0.5-0.7 mg / mL, the concentration of dithiothreitol is 0.8-0.9 mg / mL, and the concentration of ATP is 5-7 mg / mL; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride solution, phosphate buffer or 4-hydroxyethylpiperazineethanesulfonic acid solution; the concentration of the buffer component in the buffer solution is 0.03-0.05 M, and the pH of the buffer solution is 7.0-9.0; preferably, the potassium salt is potassium chloride, and the magnesium salt is magnesium chloride; The concentration of acetyl-CoA in the reagent 2 is 0.4-0.6 mg / mL; The concentration of pyruvate kinase in the reagent three is 2-4 U / mL; The concentration of lactate dehydrogenase in the reagent 4 is 2-3 U / mL; The concentration of phosphoenolpyruvate in the reagent 5 is 0.7-0.8 mg / mL; The concentration of NADH in the reagent 6 is 0.8-1.8 mg / mL; In the working solution, the volume ratio of reagent three, reagent four, reagent five, and reagent six is ​​1:1:4:4.

[0011] In the present invention, when the concentration of NADH is within the above-mentioned limit, a better detection effect can be achieved. If the concentration of NADH is too high or too low, the ACC enzyme activity measurement value in the sample to be tested will be reduced, thereby affecting the accuracy of the detection.

[0012] In addition, the present invention has found that if Mg is not added during detection 2+ It will also reduce the ACC enzyme activity measurement value in the sample to be tested, which is not conducive to obtaining accurate test results.

[0013] In the present invention, the specific types of potassium salts and magnesium salts are not limited, as long as they can provide potassium and magnesium ions to the system and do not affect the reaction of the substrate.

[0014] Preferably, in the reagent 1, the concentration of potassium salt is 1.6 mg / mL, the concentration of magnesium salt is 0.86-0.87 mg / mL, the concentration of NaHCO3 is 0.6 mg / mL, the concentration of dithiothreitol is 0.83-0.84 mg / mL, and the concentration of ATP is 6 mg / mL; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride solution; the concentration of the buffer component in the buffer solution is 0.04 M, and the pH of the buffer solution is 8.0; The concentration of acetyl-CoA in the reagent 2 is 0.5 mg / mL; The concentration of pyruvate kinase in the reagent three is 3U / mL; The concentration of lactate dehydrogenase in the reagent 4 is 2.7-2.8 U / mL; The concentration of phosphoenolpyruvate in the reagent 5 is 0.74-0.75 mg / mL; The concentration of NADH in the reagent 6 is 0.86 mg / mL.

[0015] In the method of the present invention, in the first reaction system, the volume ratio of the sample to be tested to reagent one, working solution, and reagent two is 1:5:10:4; In the second reaction system, the volume ratio of distilled water to reagent one, working solution, and reagent two is 1:5:10:4.

[0016] The method of the present invention further includes a pre-treatment step before the sample is tested. The pre-treatment method is as follows: If the sample to be tested is solid acetyl-CoA carboxylase, it is dissolved in the extraction mixture and then used for testing; If the sample to be tested is a solution containing acetyl-CoA carboxylase, it is directly used for testing; If the sample to be tested is non-liquid animal or plant tissue, cell or bacteria, it is first extracted with the extraction mixture, and then the supernatant obtained by centrifugation is used for testing; The extraction mixture comprises extraction solution 1 and extraction solution 2, wherein the extraction solution 1 comprises: buffer solution, disodium ethylenediaminetetraacetate, ascorbic acid, dithiothreitol, polyvinyl pyrrolidone, bovine serum albumin, glycerol and concentrated hydrochloric acid; the extraction solution 2 comprises protease inhibitors and water.

[0017] The first extracting solution of the present invention can be used to extract ACC in the sample to be tested. The second extracting solution can achieve the inhibition of protease.

[0018] In the extracting solution 1 of the method of the present invention, the concentration of disodium ethylenediaminetetraacetate is 0.1-0.4 mg / mL, the concentration of ascorbic acid is 0.3-0.5 mg / mL, the concentration of dithiothreitol is 2.5-3.5 mg / mL, the concentration of polyvinylpyrrolidone is 4-6 mg / mL, the concentration of bovine serum albumin is 0.8-1.2 mg / mL, the concentration of glycerol is 8-12%, and the concentration of concentrated hydrochloric acid is 0.4-0.5%; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride solution, phosphate buffer or 4-hydroxyethylpiperazineethanesulfonic acid solution; the concentration of the buffer component in the extracting solution 1 is 0.08-0.12 M; The protease inhibitor in the second extract is phenylmethylsulfonyl fluoride, and the concentration of phenylmethylsulfonyl fluoride is 15-18 mg / mL; The volume ratio of the extract 1 to the extract 2 is 99:1; Preferably, in the extract solution 1, the concentration of disodium ethylenediaminetetraacetate is 0.2-0.3 mg / mL, the concentration of ascorbic acid is 0.4 m / mL, the concentration of dithiothreitol is 3.08-3.1 mg / mL, the concentration of polyvinyl pyrrolidone is 5 mg / mL, the concentration of bovine serum albumin is 1 mg / mL, the concentration of glycerol is 10%, and the concentration of concentrated hydrochloric acid is 0.48-0.5%; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride) solution; the concentration of the buffer component is 0.1 M; The protease inhibitor in the second extract is phenylmethylsulfonyl fluoride, and the concentration of phenylmethylsulfonyl fluoride is 16-17 mg / mL.

[0019] In the method of the present invention, the solution containing acetyl-CoA carboxylase is serum or plasma; And / or, the method of first extracting with the extraction mixture and then obtaining the supernatant by centrifugation is: mixing the sample to be tested with the extraction mixture, homogenizing in an ice bath, and centrifuging to obtain the supernatant.

[0020] In the method of the present invention, the activity of acetyl-CoA carboxylase in the sample to be tested is calculated as follows: Enzyme activity definition: Each milligram of protein in the sample to be tested catalyzes the conversion of 1 nmol NADH into 1 nmol NAD per minute in the reaction system. + is one unit of enzyme activity; ACC activity, U / mg prot=[ ×V total ÷ (ɛ×d) × 10 9 ]÷(Cpr×V sample)÷T; Where V is the volume of the first reaction system, L; ɛ: NADH molar extinction coefficient, 6.22×10 3 L / mol / cm; d: light path of the detection container, cm; Cpr: protein concentration in the sample to be tested, mg / mL; Vsample: volume of the sample to be tested in the first reaction system, mL; T: reaction time, min.

[0021] The present invention also provides a kit for detecting the activity of acetyl-CoA carboxylase, which comprises: a reagent 1, a working solution and a reagent 2, or further comprises an extraction mixed solution; The reagent 1, working solution and reagent 2 are as described above; the extraction mixed solution is as described above.

[0022] The present invention also provides the application of the above method or kit in any of the following aspects: (1) In fatty acid synthesis, understand the fatty acid synthesis rate and oil content; (2) Evaluate the inhibitory effect of anticancer drugs on fatty acid synthesis; (3) Understand the activity of fatty acid synthesis in plants; (4) Understand the activity of microorganisms that rely on acetyl-CoA carboxylase to synthesize secondary metabolites; The application is achieved by measuring the activity of acetyl-CoA carboxylase.

[0023] The beneficial effects of the present invention are at least: 1. Simplified determination process: The method of the present invention omits the complicated reagent reaction steps, thus further simplifying the determination process.

[0024] 2. Time-saving and high efficiency: The method of the present invention adopts a coupled reaction to perform determination in one step, and there is no need to prepare a standard curve, which greatly shortens the experimental time and improves the efficiency of the determination.

[0025] 3. Eliminate the influence of ATP: In the traditional method, ATP and phosphorus determination reagent will react, affecting the accuracy of the experimental results, while the method of the present invention can more accurately reflect the activity of ACC.

[0026] 4. Eliminate the influence of excessive inorganic phosphorus in the sample: In the traditional method, excessive inorganic phosphorus in the sample will have a greater impact on the experimental results, while the method of the present invention is not affected by the inorganic phosphorus content in the sample, thereby improving the accuracy of the determination.

[0027] 5. The method of the present invention does not require a two-step reaction and can detect the progress of the enzymatic reaction in real time, and can be used to study the enzymatic reaction kinetics of the enzyme. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0030] Example 1 This embodiment provides a kit for detecting acetyl-CoA carboxylase activity and a preparation method thereof, comprising the following steps: Extraction solution 1: Weigh 1.331 g Tris, 0.032 g disodium ethylenediaminetetraacetate, 0.044 g ascorbic acid, 0.339 g dithiothreitol, 0.55 g polyvinylpyrrolidone, 0.11 g bovine serum albumin, add 11 mL glycerol, 0.532 mL concentrated hydrochloric acid, dilute to 110 mL with water, and store at 4°C.

[0031] Extract 2: Weigh 10 mg of phenylmethylsulfonyl fluoride, add 0.6 mL of isopropanol, and store at -20°C.

[0032] Reagent 1: Weigh 9.6 mg potassium chloride, 5.2 mg magnesium chloride, 3.6 mg sodium bicarbonate, 5 mg dithiothreitol, and 0.036 g ATP, add 6 mL 0.04 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), and store at -20°C.

[0033] Reagent 2: Weigh 2.5 mg of acetyl-CoA, add 5 mL of distilled water to dissolve, and store at -20°C.

[0034] Reagent 3: Pipette 60 U of pyruvate kinase, add 20 mL of distilled water to dissolve, and store at -20°C.

[0035] Reagent 4: Absorb 55.2 U of lactate dehydrogenase, add 20 mL of distilled water to dissolve, and store at -20°C.

[0036] Reagent 5: Weigh 3.7 mg of phosphoenolpyruvic acid, add 5 mL of distilled water to dissolve, and store at -20°C.

[0037] Reagent 6: Weigh 4.3 mg of reduced coenzyme I (NADH), add 5 mL of distilled water to dissolve, and store at -20°C.

[0038] Example 2 This example provides a new method for detecting the activity of acetyl-CoA carboxylase, and compares the test results with the traditional method for simultaneously determining acetyl-CoA carboxylase with known enzyme activity.

[0039] (I) Determination of acetyl-CoA carboxylase activity by the method of the present invention: Step 1: Prepare relevant reagents according to the above Example 1.

[0040] Step 2: Prepare the extraction mixture according to the sample volume at the ratio of Extraction Solution 1: Extraction Solution 2 = 990μL: 10μL; prepare the working solution according to the ratio of Reagent 3: Reagent 4: Reagent 5: Reagent 6 = 50μL: 50μL: 200μL: 200μL for use.

[0041] Step 3: Dilute acetyl-CoA carboxylase (ordered from Sigma, enzyme activity 20 ku / mg) with the extraction mixture into enzyme solutions with different enzyme activities of 5, 50, and 100 U / mg. Take 0.01 mL of enzyme solution and add 0.05 mL of reagent 1, 0.1 mL of working solution, and 0.04 mL of reagent 2, respectively, mix thoroughly, and immediately measure the absorbance at 340 nm after 10 seconds (A1 determination), quickly place at 37°C for accurate reaction for 10 minutes, and quickly measure the absorbance at 10 minutes and 10 seconds (A2 determination); Assay = A1 assay - A2 assay, and each sample was repeated 3 times.

[0042] Step 4: Set up blank tubes at the same time: Use distilled water instead of enzyme solution, and measure the absorbance values ​​of A1 blank and A2 blank. Blank = A1 blank - A2 blank, = Determination - Blank, thus calculating the acetyl-CoA carboxylase activity.

[0043] Step 5: Enzyme activity definition: Each milligram of protein in the sample to be tested catalyzes the conversion of 1 nmol NADH into 1 nmol NAD per minute in the reaction system. + One unit of enzyme activity.

[0044] ACC activity (U / mg prot) = [ ×V total ÷ (ɛ×d) × 10 9 ]÷(Cpr×V sample)÷T=321.5× ÷Cpr.

[0045] Where V is the total volume of the reaction system, 2×10 -4 L; ɛ: NADH molar extinction coefficient, 6.22×10 3 L / mol / cm; d: 96-well UV plate light path, 0.6 cm; V sample: added sample volume, 0.01 mL; T: reaction time, 10 min; Cpr: protein concentration in the sample to be tested, mg / mL; 10 9 : Unit conversion factor, 1mol=10 9 nmol.

[0046] (II) Determination of acetyl-CoA carboxylase activity by traditional phosphorus determination colorimetric method: Step 1: Traditional method for determining acetyl-CoA carboxylase, the reagents include: Extraction solution 1: Weigh 1.331 g Tris, 0.032 g disodium ethylenediaminetetraacetate, 0.044 g ascorbic acid, 0.339 g dithiothreitol, and 0.55 g polyvinylpyrrolidone, add 0.532 mL concentrated hydrochloric acid, dilute to 110 mL, and store at 4°C.

[0047] Extract 2: Weigh 10 mg of phenylmethylsulfonyl fluoride, add 0.6 mL of isopropanol, and store at -20°C.

[0048] Reagent 1: Take 7mL of 0.04mol / L hydroxymethylaminomethane hydrochloride solution (pH 8.0) and store at 4℃.

[0049] Reagent II A: Weigh 4.8 mg potassium chloride, 2.6 mg magnesium chloride, 1.8 mg sodium bicarbonate, add 3 mL 0.04 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), and store at 4°C.

[0050] Reagent II B: Weigh 0.018 g ATP and store at -20°C.

[0051] Reagent II C: Weigh 2.5 mg of dithiothreitol and store at -20°C.

[0052] Reagent 3: Weigh 1 mg of acetyl-CoA and store at -20°C.

[0053] Reagent 4: Weigh 0.5 g ascorbic acid and store at 4°C.

[0054] Reagent 5: Weigh 125 mg of ammonium molybdate and store at 4°C.

[0055] Reagent 6: Take 5 mL of 2.9 mol / L sulfuric acid and store at RT.

[0056] Standard: Weigh 3.6 mg of disodium hydrogen phosphate and add 1 mL of distilled water.

[0057] Step 2: Prepare an extraction mixture according to the sample volume at a ratio of Extraction Solution 1: Extraction Solution 2 = 990 μL: 10 μL; pour all of Reagent 2 B and Reagent 2 C into Reagent 2 A to fully dissolve to obtain Reagent 2 solution; add 2 mL of distilled water to Reagent 3 and fully dissolve to obtain Reagent 3 solution; add 5 mL of distilled water to Reagent 4 and fully dissolve to obtain Reagent 4 solution; add 5 mL of distilled water to Reagent 5 and fully dissolve to obtain Reagent 5 solution; prepare the working solution according to the sample volume at a volume ratio of distilled water: Reagent 4 solution: Reagent 5 solution: Reagent 6 solution = 2:1:1:1 and use it.

[0058] Step 3: Dilute the standard sodium hydrogen phosphate with the extraction mixture into 1.25, 0.625, 0.3125, 0.15625, and 0.078 μmol / mL standards. Take 0.02 mL of the standard respectively, add 0.18 mL of the working solution and mix thoroughly. React at 37°C for 30 min, cool to room temperature, and measure the absorbance at 660 nm. Draw a standard curve with the concentration of each standard solution as the x-axis and the corresponding absorbance as the y-axis to obtain the standard equation y=kx+b.

[0059] Step 4: Dilute acetyl-CoA carboxylase (ordered from Sigma) with the extraction mixture into enzyme solutions with different enzyme activities of 5, 50, and 100 U / mg. Take 0.01 mL of enzyme solution, add 0.05 mL of reagent 2 solution and 0.04 mL of reagent 3 solution, mix well, react at 37°C for 30 minutes, boil in water bath for 5 minutes, cool to room temperature, centrifuge at 10000g for 5 minutes at room temperature, take 0.02 mL of supernatant, add 0.18 mL of working solution, mix well, react at 37°C for 30 minutes, cool to room temperature, and measure the absorbance A at 660 nm. Repeat 3 times for each sample.

[0060] Step 5: Set up control tubes at the same time: Take 0.01mL enzyme solution, add 0.04mL reagent 1 and 0.05mL reagent 2 solution, mix thoroughly, react at 37℃ for 30min, boil in water bath for 5min, cool to room temperature, centrifuge at 10000g for 5min at room temperature, take 0.02mL supernatant and add 0.18mL working solution, mix thoroughly, react at 37℃ for 30min, cool to room temperature, measure the absorbance value A control at 660nm, and repeat 3 times for each sample. Substitute the difference between the two absorbances (A determination - A control) into the standard curve in step 3 above to obtain x, and then calculate the activity of acetyl-CoA carboxylase.

[0061] Step 6: Definition of enzyme activity: One ACC activity unit is the amount of 1 μmol of inorganic phosphorus produced per mg of protein in the sample to be tested per hour.

[0062] ACC enzyme activity (U / mg prot) = x × Vtotal ÷ (Vsample × Cpr) ÷ T = 20x ÷ Cpr.

[0063] Where Vtotal: total volume of enzymatic reaction, 0.1 mL; Vsample: added sample volume, 0.01 mL; T: reaction time, 0.5 h; Cpr: sample protein concentration, mg / mL.

[0064] The experimental results of the two methods are shown in Table 1.

[0065] Table 1

[0066] As shown in Table 1, when the method of the present invention is used to detect acetyl-CoA carboxylase with different enzyme activities, the calculated enzyme activity has no significant difference and is equivalent to the actual activity, the relative error is within ±8%, the coefficient of variation is within 5%, and the precision is good; when the acetyl-CoA carboxylase is detected by the traditional phosphorus determination colorimetric method, the calculated result is very different from the true value: when the enzyme activity is low, the amount of ATP consumed by the determination tube is very small, and the content of phosphorus generated is also very small. At the same time, the control tube does not undergo an enzymatic reaction (no substrate acetyl-CoA is added), and the phosphorus content does not change, resulting in the determination of the phosphorus value of the determination tube and the control tube being almost the same. At this time, the result of the determination at low enzyme activity is almost 0, so the traditional method is not accurate for detecting samples with low enzyme activity. Theoretically, the ATP added to the test tube and the control tube will both develop color. When the ACC enzyme activity is large, most of the substrate ATP in the test tube is consumed, that is, the absorbance value = (generated phosphorus color value + sample background value), the ATP in the control tube is not consumed but will produce an absorbance value, so the absorbance value of the control tube = (ATP color value + sample background value), so (test tube absorbance value - control tube absorbance value) = (generated phosphorus color value - ATP color value), the test results are all small. In summary, the detection method of the present invention can more truly determine the activity of acetyl-CoA carboxylase, and has good accuracy and precision.

[0067] Example 3 This embodiment further provides an application of the kit for detecting acetyl-CoA carboxylase activity of the present invention, comprising the following steps: Step 1: Prepare relevant reagents according to the above Example 1.

[0068] Step 2: Prepare the extraction mixture according to the sample volume at the ratio of Extraction Solution 1: Extraction Solution 2 = 990μL: 10μL; prepare the working solution according to the ratio of Reagent 3: Reagent 4: Reagent 5: Reagent 6 = 50μL: 50μL: 200μL: 200μL for use.

[0069] Step 3: Weigh 2 portions of 0.1g animal tissue, 2 portions of 0.1g plant tissue, and 2 cells, add 1mL of extraction mixture to each, homogenize in an ice bath, centrifuge and take the supernatant for testing. Then take 2 serum samples for direct testing. Take 0.01mL of sample supernatant / serum, add 0.05mL of reagent 1, 0.1mL of working solution, and 0.04mL of reagent 2, mix thoroughly, and immediately measure the absorbance value at 340nm after 10s (A1 determination), quickly place at 37℃ for accurate reaction for 10min, and quickly measure the absorbance value at 10min10s (A2 determination); Assay = A1 assay - A2 assay, and each sample was repeated 3 times.

[0070] Step 4: Set up blank tubes at the same time: Use distilled water instead of sample supernatant / serum, and measure the absorbance values ​​of A1 blank and A2 blank. Blank = A1 blank - A2 blank, = Determination - Blank, thus calculating the acetyl-CoA carboxylase activity.

[0071] Step 5: Enzyme activity definition: Each milligram of protein in the sample to be tested catalyzes the conversion of 1 nmol NADH into 1 nmol NAD per minute in the reaction system. + One unit of enzyme activity.

[0072] ACC activity (U / mg prot) = [ ×V total ÷ (ɛ×d) × 10 9 ]÷(Cpr×V sample)÷T=321.5× ÷Cpr.

[0073] Where V is the total volume of the reaction system, 2×10 -4 L; ɛ: NADH molar extinction coefficient, 6.22×10 3 L / mol / cm; d: 96-well UV plate light path, 0.6 cm; V sample: added sample volume, 0.01 mL; T: reaction time, 10 min; Cpr: protein concentration in the sample to be tested, mg / mL; 10 9 : Unit conversion factor, 1mol=10 9 nmol.

[0074] The experimental results are shown in Table 2.

[0075] Table 2

[0076] Example 4 In this example, a spiked experiment was performed to verify the reliability of the method of the present invention and the accuracy of the extraction method.

[0077] Step 1: Prepare relevant reagents according to the above Example 1.

[0078] Step 2: Prepare the extraction mixture according to the sample volume at the ratio of Extraction Solution 1: Extraction Solution 2 = 990μL: 10μL; prepare the working solution according to the ratio of Reagent 3: Reagent 4: Reagent 5: Reagent 6 = 50μL: 50μL: 200μL: 200μL for use.

[0079] Step 3: Determine the activity of acetyl-CoA carboxylase in the sample. The experimental steps are the same as Step 3, Step 4 and Step 5 of Example 3.

[0080] Step 4: Take 3 portions of 0.1g animal tissue 1 (mouse liver), add 5, 20, 50U acetyl-CoA carboxylase (ordered by Sigma), respectively; take 3 portions of 0.1g animal tissue 2 (mouse brain tissue), add 5, 20, 50U acetyl-CoA carboxylase (ordered by Sigma), respectively; take 3 portions of 0.1g plant sample 1 (sunflower leaves), add 5, 50, 100U / mg acetyl-CoA carboxylase (ordered by Sigma), respectively; take 3 portions of 0.1g plant tissue 2 (rice leaves), add 5, 50, 100U / mg acetyl-CoA carboxylase (ordered by Sigma), and measure according to steps 3, 4, and 5 in Example 3 to calculate the spiked recovery rate.

[0081] The experimental results are shown in Table 3 below.

[0082] Table 3

[0083] As shown in Table 3, the spiked recoveries of the present invention are all within 98.3%-110.5%, indicating that the method is reliable and suitable, and can more accurately determine the acetyl-CoA carboxylase activity in the sample.

[0084] Example 5 In this example, the activity of acetyl-CoA carboxylase at different time periods was measured to verify the good stability of the method of the present invention.

[0085] Step 1: Prepare relevant reagents according to the above Example 1.

[0086] Step 2: Place extract 1, extract 2, reagent 1, reagent 2, reagent 3, reagent 4, reagent 5, and reagent 6 at 4°C, -20°C, -20°C, -20°C, -20°C, and -20°C, respectively.

[0087] Step 3: Take out all the above reagents at 0 day, 1 month, 3 months, and 6 months and conduct experiments. Conduct experiments according to the steps of Part (I) of Example 2 of the present invention, and compare the data changes at different placement times. The experimental results are shown in Table 4 below.

[0088] Table 4

[0089] As shown in Table 4, when the method of the present invention is used to detect acetyl-CoA carboxylase with different enzyme activities, the values ​​of the kit of the present invention after 0 days, 1 month, 3 months, and 6 months are not significantly different and are equivalent to the actual activity, the relative error is within ±10%, the detection value has good repeatability, and the coefficient of variation is between 1.4-7.4%. The detection kit of the present invention can be stored for at least half a year and has good stability.

[0090] Example 6 This example verifies the repeatability of the method of the present invention.

[0091] Step 1: Prepare relevant reagents according to the above Example 1.

[0092] Step 2: Intra-batch repeat experiment: Select 5U / mg and 100U / mg ACC enzymes, take 10 portions of 5U / mg ACC enzymes and 10 portions of 100U / mg ACC enzymes, and use the same method, the same reagents, the same experimental operators and the same instrument to measure the acetyl-CoA carboxylase activity every day. The experimental steps are the same as Step 2, Step 3, Step 4 and Step 5 in Part (I) of Example 2.

[0093] Step 3: Repeat the experiment between batches: Select 5U / mg and 100U / mg ACC enzymes, take 10 portions of 5U / mg ACC enzymes and 10 portions of 100U / mg ACC enzymes, and use the same method, the same experimental operators and the same instrument to continuously measure the acetyl-CoA carboxylase activity every day. The reagents used every day are freshly prepared on the same day. The experimental steps are the same as steps 2, 3, 4 and 5 in part (i) of Example 2.

[0094] The results of the intra-batch repeatability experiment are shown in Table 5 below.

[0095] Table 5

[0096] The results of the batch reproducibility experiment are shown in Table 6 below.

[0097] Table 6

[0098] As shown in Table 5 and Table 6, when the method of the present invention is used to perform intra-batch repeatability test and inter-batch repeatability test, the average value and median value for 10 days are both equivalent to the actual enzyme activity of ACC and are relatively stable; the intra-batch repeatability is less than 3%, and the inter-batch repeatability is less than 5%. This indicates that the kit of the present invention has good intra-batch repeatability and inter-batch repeatability, and high precision.

[0099] Example 7 This example verifies the effect of NADH concentration on the experimental results.

[0100] Step 1: Prepare relevant reagents according to the above Example 1.

[0101] Step 2: Prepare the extraction mixture according to the sample volume at the ratio of extraction solution 1: extraction solution 2 = 990μL: 10μL; Step 3: Prepare 3.44, 1.72, 0.86, and 0.43 mg / mL of reagent VI (NADH) respectively, and prepare the working solution according to the ratio of reagent 3: reagent 4: reagent 5: reagent VI = 50 μL: 50 μL: 200 μL: 200 μL for use.

[0102] Step 4: Take 1 animal tissue and 1 plant tissue (animal tissue: mouse liver; plant tissue: sunflower leaf), respectively, with 3.44, 1.72, 0.86, 0.43 mg / mL of reagent VI prepared as working solution, and measure according to steps 3, 4, and 5 in Example 3 to calculate the sample enzyme activity. The experimental results are shown in Table 7 below.

[0103] Table 7

[0104] This method calculates the enzyme activity by detecting the degree of NADH decrease. NADH has a characteristic absorption peak at 340nm, that is, the absorbance value at 340nm will change with the change of NADH concentration. If the NADH concentration in the formula is higher, the initial value (340nm absorbance value) will be larger, the more NADH can be consumed by the ACC enzyme, and the greater the maximum enzyme activity that can be detected; if the NADH concentration in the formula is lower, the initial value (340nm absorbance value) will be smaller, the less NADH can be consumed by the ACC enzyme, and the smaller the maximum enzyme activity that can be detected. Therefore, the initial absorbance value reflected by the NADH concentration needs to be within the reasonable range that the instrument can measure, and it needs to ensure that the consumption of NADH by most samples is sufficient.

[0105] As shown in Table 7, when the NADH concentration is between 0.86-1.72 mg / mL, the sample ACC enzyme activity is the largest, and the difference is more significant for animal samples. When the NADH concentration is greater than 3.44 mg / mL, the sample ACC enzyme activity decreases because the NADH concentration is too high, causing the absorbance value to exceed the instrument range, and the actual NADH consumption can no longer be detected; when the NADH concentration is less than 0.43 mg / mL, the sample ACC enzyme activity also shows a decreasing trend, indicating that the NADH concentration provided at this time is too low, less than the amount of NADH required by the sample ACC enzyme, so the enzyme activity is low; in general, the NADH concentration is best when it is between 0.86-1.72 mg / mL.

[0106] Example 8 In order to increase the application scope of the method of the present invention, this example uses different samples with more interfering factors (compared with commercial pure enzymes) to verify the effects of different concentrations of activators on the experimental results.

[0107] Step 1: Prepare relevant reagents according to the above Example 1.

[0108] Step 2: Prepare the extraction mixture according to the sample volume at the ratio of Extraction Solution 1: Extraction Solution 2 = 990μL: 10μL; prepare the working solution according to the ratio of Reagent 3: Reagent 4: Reagent 5: Reagent 6 = 50μL: 50μL: 200μL: 200μL for use.

[0109] Step 3: Prepare Mg 2+ :K + Reagent 1 with concentration = (0.87 mg / mL: 0 mg / mL), (0.87 mg / mL: 0.8 mg / mL), (0.87 mg / mL: 1.6 mg / mL), (0.43 mg / mL: 1.6 mg / mL), (0: ​​1.6 mg / mL) (five preparation ratios).

[0110] Step 4: Take 1 animal tissue and 1 plant tissue (animal tissue: mouse liver; plant tissue: sunflower leaf), and use reagent 1 with different ion concentration ratios to measure according to steps 3, 4, and 5 in Example 3 to calculate the sample enzyme activity. The experimental results are shown in Table 8 below.

[0111] Table 8

[0112] As shown in Table 8, when Mg 2+ :K + When the concentration is 0.87:0mg / mL, the ACC enzyme activity of the sample is normal; when Mg 2+ :K + When the concentration is 0:1.6mg / mL, the ACC enzyme activity of the sample is reduced, and the difference is more significant for the animal samples with higher enzyme activity. 2+ As an activator, it is crucial for enzymatic reactions.

[0113] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining the enzyme activity of acetyl-CoA carboxylase, characterized in that: The detection reaction process is as follows: 。 2. The method according to claim 1, characterized in that include: The sample to be tested is mixed with reagent 1, working solution and reagent 2 to obtain the first reaction system, and the absorbance value at 340 nm is detected within 30 seconds, which is counted as A1 determination; Then, the first reaction system was placed at 37±1℃ for 5-10min, and the absorbance was measured, which was calculated as A2 measurement; Determination, Determination = A1 determination - A2 determination; In addition, the sample to be tested in the first reaction system is replaced with distilled water to obtain a second reaction system, the volume of the second reaction system is the same as that of the first reaction system, and the A1 blank and A2 blank are obtained in the same manner as that of the A1 determination and the A2 determination; Calculate blank, Blank = A1 blank - A2 blank; pass Calculate the activity of acetyl-CoA carboxylase in the sample to be tested. = Determination - blank; The reagent 1 comprises: potassium salt, magnesium salt, NaHCO3, dithiothreitol, ATP and buffer solution; The second reagent includes: acetyl coenzyme A and water; The working solution includes reagent three, reagent four, reagent five, and reagent six; The reagent three includes pyruvate kinase and water; The reagent 4 includes lactate dehydrogenase and water; The reagent five includes phosphoenolpyruvic acid and water; The reagent six includes NADH and water.

3. The method according to claim 2, characterized in that In the reagent 1, the concentration of potassium salt is 0-1.6 mg / mL, the concentration of magnesium salt is 0.4-0.9 mg / mL, the concentration of NaHCO3 is 0.5-0.7 mg / mL, the concentration of dithiothreitol is 0.8-0.9 mg / mL, and the concentration of ATP is 5-7 mg / mL; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride solution, phosphate buffer or 4-hydroxyethylpiperazineethanesulfonic acid solution; the concentration of the buffer component in the buffer solution is 0.03-0.05 M, and the pH of the buffer solution is 7.0-9.0; The concentration of acetyl-CoA in the reagent 2 is 0.4-0.6 mg / mL; The concentration of pyruvate kinase in the reagent three is 2-4 U / mL; The concentration of lactate dehydrogenase in the reagent 4 is 2-3 U / mL; The concentration of phosphoenolpyruvate in the reagent 5 is 0.7-0.8 mg / mL; The concentration of NADH in the reagent 6 is 0.8-1.8 mg / mL; In the working solution, the volume ratio of reagent three, reagent four, reagent five, and reagent six is ​​1:1:4:

4.

4. The method according to claim 3, characterized in that In the reagent 1, the concentration of potassium salt is 1.6 mg / mL, the concentration of magnesium salt is 0.86-0.87 mg / mL, the concentration of NaHCO3 is 0.6 mg / mL, the concentration of dithiothreitol is 0.83-0.84 mg / mL, and the concentration of ATP is 6 mg / mL; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride solution; the concentration of the buffer component in the buffer solution is 0.04 M, and the pH of the buffer solution is 8.0; the potassium salt is potassium chloride, and the magnesium salt is magnesium chloride; The concentration of acetyl-CoA in the reagent 2 is 0.5 mg / mL; The concentration of pyruvate kinase in the reagent three is 3U / mL; The concentration of lactate dehydrogenase in the reagent 4 is 2.7-2.8 U / mL; The concentration of phosphoenolpyruvate in the reagent 5 is 0.74-0.75 mg / mL; The concentration of NADH in the reagent 6 is 0.86 mg / mL.

5. The method according to claim 3, characterized in that: In the first reaction system, the volume ratio of the sample to be tested to reagent 1, working solution, and reagent 2 is 1:5:10:4; In the second reaction system, the volume ratio of distilled water to reagent one, working solution, and reagent two is 1:5:10:

4.

6. The method according to claim 5, characterized in that Before the sample is tested, a pre-treatment step is also included, and the pre-treatment method is as follows: If the sample to be tested is solid acetyl-CoA carboxylase, it is dissolved in the extraction mixture and then used for testing; If the sample to be tested is a solution containing acetyl-CoA carboxylase, it is directly used for testing; If the sample to be tested is non-liquid animal or plant tissue, cell or bacteria, it is first extracted with the extraction mixture, and then the supernatant obtained by centrifugation is used for testing; The extraction mixture comprises extraction solution 1 and extraction solution 2, wherein the extraction solution 1 comprises: buffer solution, disodium ethylenediaminetetraacetate, ascorbic acid, dithiothreitol, polyvinyl pyrrolidone, bovine serum albumin, glycerol and concentrated hydrochloric acid; the extraction solution 2 comprises protease inhibitors and water.

7. The method according to claim 6, characterized in that In the extracting solution 1, the concentration of disodium ethylenediaminetetraacetate is 0.1-0.4 mg / mL, the concentration of ascorbic acid is 0.3-0.5 mg / mL, the concentration of dithiothreitol is 2.5-3.5 mg / mL, the concentration of polyvinylpyrrolidone is 4-6 mg / mL, the concentration of bovine serum albumin is 0.8-1.2 mg / mL, the concentration of glycerol is 8-12%, and the concentration of concentrated hydrochloric acid is 0.4-0.5%; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride solution, phosphate buffer or 4-hydroxyethylpiperazineethanesulfonic acid solution; the concentration of the buffer component in the extracting solution 1 is 0.08-0.12 M; The protease inhibitor in the second extract is phenylmethylsulfonyl fluoride, and the concentration of phenylmethylsulfonyl fluoride is 15-18 mg / mL; The volume ratio of the extract 1 to the extract 2 is 99:

1.

8. The method according to claim 7, characterized in that In the extract solution 1, the concentration of disodium ethylenediaminetetraacetate is 0.2-0.3 mg / mL, the concentration of ascorbic acid is 0.4 mg / mL, the concentration of dithiothreitol is 3.08-3.1 mg / mL, the concentration of polyvinylpyrrolidone is 5 mg / mL, the concentration of bovine serum albumin is 1 mg / mL, the concentration of glycerol is 10%, and the concentration of concentrated hydrochloric acid is 0.48-0.5%; the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride) solution; the concentration of the buffer component is 0.1 M; The protease inhibitor in the second extract is phenylmethylsulfonyl fluoride, and the concentration of phenylmethylsulfonyl fluoride is 16-17 mg / mL.

9. The method according to claim 6, characterized in that The solution containing acetyl-CoA carboxylase is serum or plasma; And / or, the method of first extracting with the extraction mixture and then obtaining the supernatant by centrifugation is: mixing the sample to be tested with the extraction mixture, homogenizing in an ice bath, and centrifuging to obtain the supernatant.

10. The method according to any one of claims 2 to 9, characterized in that: The activity of acetyl-CoA carboxylase in the sample to be tested is calculated as follows: Enzyme activity definition: Each milligram of protein in the sample to be tested catalyzes the conversion of 1 nmol NADH into 1 nmol NAD per minute in the reaction system. + is one unit of enzyme activity; ACC activity, U / mg prot=[ ×V total ÷ (ɛ×d) × 10 9 ]÷(Cpr×V sample)÷T; Where V is the volume of the first reaction system, L; ɛ: NADH molar extinction coefficient, 6.22×10 3 L / mol / cm; d: light path of the detection container, cm; Cpr: protein concentration in the sample to be tested, mg / mL; Vsample: volume of the sample to be tested in the first reaction system, mL; T: reaction time, min.

11. A kit for detecting acetyl-CoA carboxylase activity, characterized in that: include: Reagent 1, working solution and reagent 2, or further comprising an extraction mixture; The reagent 1, working solution and reagent 2 are as described in any one of claims 2-4; the extraction mixed solution is as described in any one of claims 6-8.

12. Use of the method according to any one of claims 1 to 10 or the kit according to claim 11 in any of the following aspects: (1) In fatty acid synthesis, understand the fatty acid synthesis rate and oil content; (2) Evaluate the inhibitory effect of anticancer drugs on fatty acid synthesis; (3) Understand the activity of fatty acid synthesis in plants; (4) Understand the activity of microorganisms that rely on acetyl-CoA carboxylase to synthesize secondary metabolites; The application is achieved by measuring the activity of acetyl-CoA carboxylase.

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