Enzyme Activity Assay Method, Kit and Application of Acetyl-CoA Carboxylase

Abstract

The present invention relates to the technical field of substance detection, and specifically discloses a method for determining the enzyme activity of acetyl-CoA carboxylase, a kit and an application thereof. The present invention provides a method for determining the enzyme activity of acetyl-CoA carboxylase, and its detection reaction process is as follows:. The detection method of the present invention can achieve rapid and accurate detection of ACC enzyme activity, can perform efficient ACC determination in research and applications, and has broad application prospects.

Description

Technical Field

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

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

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

[0004] However, this method has some problems: (1) One of the substrates of the enzymatic reaction, ATP, reacts with the phosphorus-determining reagent, affecting the accuracy of the experimental results; (2) There is a large amount of inorganic phosphate in animal and plant samples. If the background inorganic phosphate has exceeded 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 is low, dilution will cause unstable or even negative measurement results, affecting the accurate measurement of ACC activity; (3) This method requires two-step reactions (enzymatic reaction and phosphorus-determining reaction), which consumes a lot of time. It needs to make a standard curve and requires multiple steps for measurement, increasing 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 method for determining the enzyme activity of ACC. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a new method for accurately determining the activity of ACC.

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

[0007] 。

[0008] The present invention provides a new method for determining the activity of acetyl-CoA carboxylase (ACC). Instead of reflecting the activity of ACC by the content of inorganic phosphorus, the activity of ACC is determined through the further reaction of ADP. Specifically, the technical means for 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 the generation of NAD from NADH + , and the activity of ACC is reflected by measuring NADH at a wavelength of 340 nm.

[0009] The method of the present invention is not affected by the content of inorganic phosphorus in the sample to be tested. The experimental results are more accurate, and there is no need to prepare a standard curve. Only two detections of the sample to be tested and the blank sample are required to quickly obtain the detection result, improving the detection efficiency.

[0010] The method of the present invention includes: mixing the sample to be tested with Reagent 1, the working solution, and Reagent 2 to obtain a first reaction system, and detecting the absorbance at 340 nm within 30 s, which is recorded as A1 measurement;

[0011] Then, the first reaction system is placed at 37 ± 1 °C for reaction for 5 - 10 min, and the absorbance is measured, which is recorded as A2 measurement; calculate measurement, measurement = A1 measurement - A2 measurement;

[0012] Another second reaction system is obtained by replacing the sample to be tested in the first reaction system with distilled water. The volume of the second reaction system is the same as that of the first reaction system, and A1 blank and A2 blank are obtained in the same way as obtaining A1 measurement and A2 measurement; calculate blank, blank = A1 blank - A2 blank;

[0013] Through calculation, the activity of acetyl-CoA carboxylase in the sample to be tested is obtained, = measurement - blank;

[0014] Reagent 1 includes: potassium salt, magnesium salt, NaHCO3, dithiothreitol, ATP, and buffer solution;

[0015] Reagent 2 includes: acetyl-CoA and water;

[0016] The working solution includes Reagent 3, Reagent 4, Reagent 5, and Reagent 6;

[0017] Reagent 3 includes pyruvate kinase and water;

[0018] Reagent 4 includes lactate dehydrogenase and water;

[0019] Reagent 5 includes phosphoenolpyruvate and water;

[0020] Reagent 6 includes NADH and water.

[0021] In Reagent 1 of the method of the present invention, the concentration of the potassium salt is 0 - 1.6 mg / mL, the concentration of the 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 a tris(hydroxymethyl)aminomethane hydrochloride solution, a phosphate buffer solution, or a 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic 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;

[0022] The concentration of acetyl coenzyme A in Reagent 2 is 0.4 - 0.6 mg / mL;

[0023] The concentration of pyruvate kinase in Reagent 3 is 2 - 4 U / mL;

[0024] The concentration of lactate dehydrogenase in Reagent 4 is 2 - 3 U / mL;

[0025] The concentration of phosphoenolpyruvate in Reagent 5 is 0.7 - 0.8 mg / mL;

[0026] The concentration of NADH in Reagent 6 is 0.8 - 1.8 mg / mL;

[0027] In the working solution, the volume ratio of Reagent 3, Reagent 4, Reagent 5, and Reagent 6 is 1:1:4:4.

[0028] In the present invention, when the concentration of NADH is within the above limits, better detection effects can be achieved. If the concentration of NADH is too high or too low, the measured value of the ACC enzyme activity in the sample to be tested will be reduced, thus affecting the accuracy of the detection.

[0029] In addition, the present invention has found that if Mg is not added during the detection 2+ the measured value of the ACC enzyme activity in the sample to be tested will also be reduced, thus being unfavorable for obtaining accurate detection results.

[0030] In the present invention, the specific types of the potassium salt and the magnesium salt are not limited as long as they can provide potassium and magnesium ions for the system and do not affect the reaction of the substrate.

[0031] Preferably, in the first reagent, 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;

[0032] In the second reagent, the concentration of acetyl coenzyme A is 0.5 mg / mL;

[0033] In the third reagent, the concentration of pyruvate kinase is 3 U / mL;

[0034] In the fourth reagent, the concentration of lactate dehydrogenase is 2.7 - 2.8 U / mL;

[0035] In the fifth reagent, the concentration of phosphoenolpyruvate is 0.74 - 0.75 mg / mL;

[0036] In the sixth reagent, the concentration of NADH is 0.86 mg / mL.

[0037] In the method of the present invention, in the first reaction system, the volume ratio of the test sample, the first reagent, the working solution, and the second reagent is 1:5:10:4;

[0038] In the second reaction system, the volume ratio of distilled water, the first reagent, the working solution, and the second reagent is 1:5:10:4.

[0039] In the method of the present invention, before detecting the test sample, a pretreatment step is further included, and the pretreatment method is as follows:

[0040] If the test sample is solid acetyl coenzyme A carboxylase, it is dissolved in the extraction mixture for detection;

[0041] If the test sample is a solution containing acetyl coenzyme A carboxylase, it is directly used for detection;

[0042] If the test sample is a non-liquid animal or plant tissue, cell, or bacterium, it is first extracted with the extraction mixture, and then the supernatant obtained by centrifugation is taken for detection;

[0043] The extraction mixture includes extraction solution 1 and extraction solution 2. Extraction solution 1 includes: buffer solution, disodium ethylenediaminetetraacetate, ascorbic acid, dithiothreitol, polyvinylpyrrolidone, bovine serum albumin, glycerol, and concentrated hydrochloric acid; extraction solution 2 includes protease inhibitor and water.

[0044] The extraction solution 1 of the present invention can be used to extract ACC from the test sample. Extraction solution 2 can achieve protease inhibition.

[0045] In the extraction solution I 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 a tris(hydroxymethyl)aminomethane hydrochloride solution, a phosphate buffer solution, or a 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid solution; the concentration of the buffer component in the extraction solution I is 0.08 - 0.12 M;

[0046] The protease inhibitor in the extraction solution II is phenylmethylsulfonyl fluoride, and the concentration of phenylmethylsulfonyl fluoride is 15 - 18 mg / mL;

[0047] The volume ratio of the extraction solution I to the extraction solution II is 99:1;

[0048] Preferably, in the extraction solution I, 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 a tris(hydroxymethyl)aminomethane hydrochloride solution; the concentration of the buffer component is 0.1 M;

[0049] The protease inhibitor in the extraction solution II is phenylmethylsulfonyl fluoride, and the concentration of phenylmethylsulfonyl fluoride is 16 - 17 mg / mL.

[0050] In the method of the present invention, the solution containing acetyl-CoA carboxylase is serum or plasma;

[0051] And / or, the method of first extracting with an extraction mixture and then taking the supernatant obtained by centrifugation is: mixing the sample to be tested with the extraction mixture and then homogenizing in an ice bath, and centrifuging to obtain the supernatant.

[0052] In the method of the present invention, the activity of acetyl-CoA carboxylase in the sample to be tested is calculated as follows:

[0053] Enzyme activity definition: Each milligram of protein in the sample to be tested catalyzes the conversion of 1 nmol of NADH to 1 nmol of NAD per minute in the reaction system + as one enzyme activity unit;

[0054] ACC activity, U / mg prot = × Vtotal reaction ÷ (ɛ × d) × 10 9÷ (Cpr × V_sample) ÷ T;

[0055] Wherein, V_total_reverse is the volume of the first reaction system, L; ɛ: molar extinction coefficient of NADH, 6.22×10 3 L / mol / cm; d: optical path of the detection container, cm; Cpr: protein concentration in the sample to be measured, mg / mL; V_sample: volume of the sample to be measured in the first reaction system, mL; T: reaction time, min.

[0056] The present invention also provides a kit for detecting the activity of acetyl-CoA carboxylase, which includes: reagent one, working solution and reagent two, or further includes an extraction mixture;

[0057] The reagent one, working solution and reagent two are as described above; the extraction mixture is as described above.

[0058] The present invention also provides the application of the above method or kit in any of the following aspects:

[0059] (1) In fatty acid synthesis, to understand the fatty acid synthesis rate and the level of oil content;

[0060] (2) To evaluate the inhibitory effect of anti-tumor drugs on fatty acid synthesis;

[0061] (3) To understand the activity degree of fatty acid synthesis in plants;

[0062] (4) To understand the activity degree of microorganisms that synthesize secondary metabolites depending on acetyl-CoA carboxylase;

[0063] The above application is achieved by measuring the activity of acetyl-CoA carboxylase.

[0064] The beneficial effects of the present invention are at least as follows:

[0065] 1. Simplify the measurement process: The method of the present invention omits the complex reagent reaction steps, making the measurement process more simplified.

[0066] 2. Time-saving and efficient: The method of the present invention uses a coupled reaction for one-step measurement, without the need to prepare a standard curve, greatly shortening the experimental time and improving the measurement efficiency.

[0067] 3. Eliminate the influence of ATP: In the traditional method, ATP and the phosphorus determination reagent will react, affecting the accuracy of the experimental results, while the method of the present invention can more accurately reflect the ACC activity.

[0068] 4. Eliminate the influence of more inorganic phosphorus in the sample: In the traditional method, more 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, improving the measurement accuracy.

[0069] 5. The method of the present invention does not require two-step reactions and can detect the progress of enzymatic reactions in real time, and is used for exploring the enzymatic reaction kinetics of this enzyme. Detailed implementation manners

[0070] The preferred implementation manners of the present invention will be described in detail below in conjunction with embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0071] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels or prepared by conventional methods in the art.

[0072] Embodiment 1

[0073] This embodiment provides a kit for detecting the activity of acetyl-CoA carboxylase and its preparation method, including the following steps:

[0074] Extract 1: Weigh 1.331 g of Tris, 0.032 g of disodium ethylenediaminetetraacetate, 0.044 g of ascorbic acid, 0.339 g of dithiothreitol, 0.55 g of polyvinylpyrrolidone, 0.11 g of bovine serum albumin, add 11 mL of glycerol, 0.532 mL of concentrated hydrochloric acid, and make up the volume to 110 mL with water, and store at 4 °C.

[0075] Extract 2: Weigh 10 mg of phenylmethylsulfonyl fluoride and add it to 0.6 mL of isopropanol, and store at -20 °C.

[0076] Reagent 1: Weigh 9.6 mg of potassium chloride, 5.2 mg of magnesium chloride, 3.6 mg of sodium bicarbonate, 5 mg of dithiothreitol, 0.036 g of ATP, add them to 6 mL of 0.04 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), and store at -20 °C.

[0077] Reagent 2: Weigh 2.5 mg of acetyl-CoA, add it to 5 mL of distilled water to dissolve, and store at -20 °C.

[0078] Reagent 3: Pipette 60 U of pyruvate kinase, add it to 20 mL of distilled water to dissolve, and store at -20 °C.

[0079] Reagent 4: Pipette 55.2 U of lactate dehydrogenase, add it to 20 mL of distilled water to dissolve, and store at -20 °C.

[0080] Reagent 5: Weigh 3.7 mg of phosphoenolpyruvate, add it to 5 mL of distilled water to dissolve, and store at -20 °C.

[0081] Reagent VI: Weigh 4.3 mg of nicotinamide adenine dinucleotide reduced (NADH), add 5 mL of distilled water to dissolve it, and store it at -20 °C.

[0082] Example 2

[0083] This example provides a new method for detecting the activity of acetyl-CoA carboxylase, and the comparison of test effects is carried out by simultaneously measuring the acetyl-CoA carboxylase with known enzyme activity by this method and the traditional method.

[0084] (1) Measuring the activity of acetyl-CoA carboxylase by the method of the present invention:

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

[0086] Step 2: Prepare the extraction mixture according to the ratio of extraction solution I: extraction solution II = 990 μL: 10 μL according to the sample volume; prepare the working solution according to the ratio of reagent III: reagent IV: reagent V: reagent VI = 50 μL: 50 μL: 200 μL: 200 μL and then use it.

[0087] Step 3: Dilute the 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. Respectively take 0.01 mL of the enzyme solution and add 0.05 mL of reagent I, 0.1 mL of the working solution, and 0.04 mL of reagent II, mix well, immediately measure the absorbance value A1 at 340 nm at 10 s, quickly place it at 37 °C for an accurate reaction for 10 min, and quickly measure the absorbance value A2 at 10 min 10 s; Measurement = A1 measurement - A2 measurement, and each sample is repeated 3 times.

[0088] Step 4: At the same time, set up a blank tube: replace the enzyme solution with distilled water, and measure the absorbance values A1 blank and A2 blank, Blank = A1 blank - A2 blank, = Measurement - Blank, so as to calculate the activity of acetyl-CoA carboxylase.

[0089] Step 5:

[0090] Definition of enzyme activity: Each milligram of protein in the sample to be measured catalyzes the conversion of 1 nmol of NADH to 1 nmol of NAD in the reaction system per minute + is an enzyme activity unit.

[0091] ACC activity (U / mg prot) = ×V total reaction ÷ (ɛ × d) × 10 9 ÷ (Cpr × V sample) ÷ T = 321.5 × ÷Cpr.

[0092] Wherein V_total_reverse is the total volume of the reaction system, 2×10 -4 L; ɛ: the molar extinction coefficient of NADH, 6.22×10 3 L / mol / cm; d: the optical path of the 96-well UV plate, 0.6 cm; V_sample: the volume of the added sample, 0.01 mL; T: the reaction time, 10 min; Cpr: the protein concentration in the sample to be measured, mg / mL; 10 9 : the unit conversion coefficient, 1 mol = 10 9 nmol.

[0093] (2) Determining the activity of acetyl-CoA carboxylase by the traditional phosphorus determination colorimetric method:

[0094] Step 1: Determining acetyl-CoA carboxylase by the traditional method. The reagents include:

[0095] Extract 1: Weigh 1.331 g of Tris, 0.032 g of disodium ethylenediaminetetraacetate, 0.044 g of ascorbic acid, 0.339 g of dithiothreitol, 0.55 g of polyvinylpyrrolidone, add 0.532 mL of concentrated hydrochloric acid, and make up the volume to 110 mL, store at 4°C.

[0096] Extract 2: Weigh 10 mg of benzylsulfonyl fluoride and add it to 0.6 mL of isopropanol, store at -20°C.

[0097] Reagent 1: Take 7 mL of 0.04 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), store at 4°C.

[0098] Reagent 2A: Weigh 4.8 mg of potassium chloride, 2.6 mg of magnesium chloride, 1.8 mg of sodium bicarbonate, add them to 3 mL of 0.04 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), store at 4°C.

[0099] Reagent 2B: Weigh 0.018 g of ATP, store at -20°C.

[0100] Reagent 2C: Weigh 2.5 mg of dithiothreitol, store at -20°C.

[0101] Reagent 3: Weigh 1 mg of acetyl-CoA, store at -20°C.

[0102] Reagent 4: Weigh 0.5 g of ascorbic acid, store at 4°C.

[0103] Reagent 5: Weigh 125 mg of ammonium molybdate, store at 4°C.

[0104] Reagent 6: Take 5 mL of 2.9 mol / L sulfuric acid, store at RT.

[0105] Standard: Weigh 3.6 mg of disodium hydrogen phosphate and add 1 mL of distilled water.

[0106] Step 2: Prepare the extraction mixture according to the ratio of Extraction Solution 1: Extraction Solution 2 = 990 μL: 10 μL based on the sample volume; pour all of Reagent 2B and Reagent 2C into Reagent 2A and dissolve thoroughly to obtain Reagent 2 solution; add 2 mL of distilled water to Reagent 3 and dissolve thoroughly to obtain Reagent 3 solution; add 5 mL of distilled water to Reagent 4 and dissolve thoroughly to obtain Reagent 4 solution; add 5 mL of distilled water to Reagent 5 and dissolve thoroughly to obtain Reagent 5 solution; prepare the working solution according to the volume ratio of distilled water: Reagent 4 solution: Reagent 5 solution: Reagent 6 solution = 2: 1: 1: 1 based on the sample volume and then use it.

[0107] Step 3: Dilute the standard disodium hydrogen phosphate with the extraction mixture to standards of 1.25, 0.625, 0.3125, 0.15625, 0.078 μmol / mL. Take 0.02 mL of each standard respectively, add 0.18 mL of the working solution and mix well. React at 37 °C for 30 min, cool to room temperature, and measure the absorbance at 660 nm. Using the concentration of each standard solution as the x-axis and its corresponding absorbance as the y-axis, plot the standard curve to obtain the standard equation y = kx + b.

[0108] Step 4: Dilute acetyl-CoA carboxylase (ordered from sigma company) with the extraction mixture to enzyme solutions with different enzyme activities of 5, 50, 100 U / mg. Take 0.01 mL of each enzyme solution respectively, add 0.05 mL of Reagent 2 solution and 0.04 mL of Reagent 3 solution, mix well, react at 37 °C for 30 min, boil in a water bath for 5 min, cool to room temperature, centrifuge at 10000 g at room temperature for 5 min, take 0.02 mL of the supernatant, add 0.18 mL of the working solution and mix well, react at 37 °C for 30 min, cool to room temperature, and measure the absorbance A measurement at 660 nm. Repeat each sample 3 times.

[0109] Step 5: Set up control tubes simultaneously: Take 0.01 mL of each enzyme solution respectively, add 0.04 mL of Reagent 1 and 0.05 mL of Reagent 2 solution, mix well, react at 37 °C for 30 min, boil in a water bath for 5 min, cool to room temperature, centrifuge at 10000 g at room temperature for 5 min, take 0.02 mL of the supernatant, add 0.18 mL of the working solution and mix well, react at 37 °C for 30 min, cool to room temperature, and measure the absorbance A control at 660 nm. Repeat each sample 3 times. Calculate the acetyl-CoA carboxylase activity by substituting the difference in the two absorbances (A measurement - A control) into the standard curve in Step 3 above to obtain x.

[0110] Step 6:

[0111] Enzyme activity definition: One ACC activity unit is the amount of protein in each milligram of the sample to be tested that produces 1 μmol of inorganic phosphorus per hour.

[0112] ACC enzyme activity (U / mg prot) = x × Vtotal ÷ (Vsample × Cpr) ÷ T = 20x ÷ Cpr.

[0113] Where Vtotal: total volume of the enzymatic reaction, 0.1 mL; Vsample: volume of the added sample, 0.01 mL; T: reaction time, 0.5 h; Cpr: sample protein concentration, mg / mL.

[0114] The experimental results of the two testing methods are shown in Table 1.

[0115] Table 1

[0116]

[0117] As shown in Table 1, when the acetyl-CoA carboxylase with different enzyme activities is detected by the method of the present invention, the calculated enzyme activities have no significant difference and are equivalent to the actual activities, with a relative error within ±8% and a coefficient of variation within 5%, indicating good precision. When the acetyl-CoA carboxylase is detected by the traditional phosphorus determination and color development method, the calculated results are very different from the true values: when the enzyme activity is low, since the amount of ATP consumed by the assay tube is very small, the amount of generated phosphorus is also very small. At the same time, no enzymatic reaction occurs in the control tube (substrate acetyl-CoA is not added), so the phosphorus content remains unchanged, resulting in the phosphorus determination value in the assay tube being almost the same as that in the control tube. At this time, the result measured at low enzyme activity is almost 0, so the traditional method is not accurate for detecting samples with low enzyme activity. Theoretically, the added ATP in both the assay tube and the control tube will show color. When the ACC enzyme activity is large, most of the substrate ATP in the assay tube is consumed, that is, absorbance value = (color development value of generated phosphorus + sample background value), and the ATP in the control tube is not consumed but will generate an absorbance value, so the absorbance value of the control tube = (color development value of ATP + sample background value). Therefore, (absorbance value of the assay tube - absorbance value of the control tube) = (color development value of generated phosphorus - color development value of ATP), and the measurement results are all on the small side. Generally speaking, the detection method of the present invention can more accurately measure the activity of acetyl-CoA carboxylase and has good accuracy and precision.

[0118] Example 3

[0119] This example further provides the application of the kit for detecting the activity of acetyl-CoA carboxylase of the present invention, including the following steps:

[0120] Step 1: Prepare relevant reagents according to Example 1 above.

[0121] Step 2: Prepare the extraction mixture according to the ratio of extraction solution 1: extraction solution 2 = 990 μL: 10 μL based on the sample volume; after preparing the working solution according to the ratio of reagent 3: reagent 4: reagent 5: reagent 6 = 50 μL: 50 μL: 200 μL: 200 μL, use it.

[0122] Step 3: Weigh 2 portions of 0.1 g of animal tissue, 2 portions of 0.1 g of plant tissue, and 2 cells, add 1 mL of the extraction mixture to each, homogenize in an ice bath, centrifuge, and take the supernatant for testing. Then directly test 2 serum samples. Take 0.01 mL of the sample supernatant / serum and add 0.05 mL of reagent 1, 0.1 mL of the working solution, and 0.04 mL of reagent 2, mix well, immediately measure the absorbance value A1 at 340 nm at 10 s, quickly place it at 37 °C for an accurate reaction for 10 min, and quickly measure the absorbance value A2 at 10 min 10 s; Measurement = A1 measurement - A2 measurement, and each sample is repeated 3 times.

[0123] Step 4: At the same time, set up a blank tube: replace the sample supernatant / serum with distilled water, and measure the absorbance values A1 blank and A2 blank. Blank = A1 blank - A2 blank. = Measurement - Blank, thereby calculating the activity of acetyl-CoA carboxylase.

[0124] Step 5:

[0125] Enzyme activity definition: Each milligram of protein in the sample to be tested catalyzes the conversion of 1 nmol of NADH to 1 nmol of NAD in the reaction system per minute. + is one enzyme activity unit.

[0126] ACC activity (U / mg prot) = ×V total reaction ÷ (ɛ × d) × 10 9 ÷ (Cpr × V sample) ÷ T = 321.5 × ÷ Cpr.

[0127] Among them, V total reaction is the total volume of the reaction system, 2 × 10 -4 L; ɛ: molar extinction coefficient of NADH, 6.22 × 10 3 L / mol / cm; d: optical path of the 96-well UV plate, 0.6 cm; V sample: volume of the sample added, 0.01 mL; T: reaction time, 10 min; Cpr: protein concentration in the sample to be tested, mg / mL; 10 9 : unit conversion coefficient, 1 mol = 10 9 nmol.

[0128] The experimental results are shown in Table 2.

[0129] Table 2

[0130]

[0131] Example 4

[0132] In this example, a spiking experiment was conducted to verify the reliability of the method of the present invention and the accuracy of the extraction method.

[0133] Step 1: Prepare relevant reagents according to Example 1 above.

[0134] Step 2: Prepare an extraction mixture according to the ratio of extraction solution 1:extraction solution 2 = 990 μL:10 μL according to the sample volume; after preparing a working solution according to the ratio of reagent three:reagent four:reagent five:reagent six = 50 μL:50 μL:200 μL:200 μL, use it.

[0135] Step 3: Measure the activity of acetyl-CoA carboxylase present in the sample itself. The experimental steps are the same as steps three, four, and five of Example 3.

[0136] Step 4: Take 3 portions of 0.1 g of animal tissue 1 (mouse liver) and add 5, 20, and 50 U of acetyl-CoA carboxylase (purchased from Sigma) respectively; 3 portions of 0.1 g of animal tissue 2 (mouse brain tissue) and add 5, 20, and 50 U of acetyl-CoA carboxylase (purchased from Sigma) respectively; 3 portions of 0.1 g of plant sample 1 (sunflower leaves) and add 5, 50, and 100 U / mg of acetyl-CoA carboxylase (purchased from Sigma) respectively; 3 portions of 0.1 g of plant tissue 2 (rice leaves) and add 5, 50, and 100 U / mg of acetyl-CoA carboxylase (purchased from Sigma) respectively, and measure according to steps three, four, and five in Example 3 to calculate the spiking recovery rate.

[0137] The experimental results are shown in Table 3 below.

[0138] Table 3

[0139]

[0140] As shown in Table 3, the spiking recovery rates of the present invention are all within 98.3% - 110.5%, indicating that the method is reliable and suitable and can accurately measure the activity of acetyl-CoA carboxylase in the sample.

[0141] Example 5

[0142] 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.

[0143] Step 1: Prepare relevant reagents according to Example 1 above.

[0144] 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, -20°C respectively.

[0145] Step 3: Take out all the above reagents at time points of 0 day, 1 month, 3 months, and 6 months and conduct experiments. Conduct experiments according to the steps in Part (1) of Example 2 of the present invention, and compare the data changes at different storage times. The experimental results are shown in Table 4 below.

[0146] Table 4

[0147]

[0148] As shown in Table 4, when detecting acetyl-CoA carboxylase with different enzyme activities by the method of the present invention, the values of the kit of the present invention at 0 day, 1 month, 3 months, and 6 months have no significant differences and are equivalent to the actual activities, with a relative error within ±10%. The detected values have 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.

[0149] Example 6

[0150] This example verifies the repeatability of the method of the present invention.

[0151] Step 1: Prepare relevant reagents according to Example 1 above.

[0152] Step 2: Intra-assay repeat experiment: Select 5 U / mg and 100 U / mg ACC enzymes. Take 10 portions of 5 U / mg ACC enzyme and 10 portions of 100 U / mg ACC enzyme, and continuously measure the activity of acetyl-CoA carboxylase every day using the same method, the same reagents, the same experimental operator, and the same instrument. The experimental steps are the same as Steps 2, 3, 4, and 5 in Part (1) of Example 2.

[0153] Step 3: Inter-assay repeat experiment: Select 5 U / mg and 100 U / mg ACC enzymes. Take 10 portions of 5 U / mg ACC enzyme and 10 portions of 100 U / mg ACC enzyme, and continuously measure the activity of acetyl-CoA carboxylase every day using the same method, the same experimental operator, and the same instrument. 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 (1) of Example 2.

[0154] The results of the intra-assay repeat experiment are shown in Table 5 below.

[0155] Table 5

[0156]

[0157] The results of the between-batch repeatability experiment are shown in Table 6 below.

[0158] Table 6

[0159]

[0160] As shown in Table 5 and Table 6, when the within-batch repeatability test and the between-batch repeatability test are carried out by the method of the present invention, the average value and the median value in 10 days are both equivalent to the actual enzyme activity of ACC and are relatively stable; the within-batch repeatability is less than 3%, and the between-batch repeatability is less than 5%. It shows that the kit of the present invention has good within-batch repeatability and between-batch repeatability and high precision.

[0161] Example 7

[0162] This example verifies the influence of the NADH concentration on the experimental results.

[0163] Step 1: Prepare the relevant reagents according to Example 1 above.

[0164] Step 2: Prepare an extraction mixture according to the ratio of extraction solution 1: extraction solution 2 = 990 μL: 10 μL according to the sample volume;

[0165] Step 3: Prepare reagent 6 (NADH) at 3.44, 1.72, 0.86, and 0.43 mg / mL respectively, and prepare a working solution according to the ratio of reagent 3: reagent 4: reagent 5: reagent 6 = 50 μL: 50 μL: 200 μL: 200 μL for use.

[0166] Step 4: Take 1 portion of animal tissue and 1 portion of plant tissue (animal tissue: mouse liver; plant tissue: sunflower leaf), and use the working solution prepared with reagent 6 at 3.44, 1.72, 0.86, and 0.43 mg / mL respectively, 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.

[0167] Table 7

[0168]

[0169] This method calculates the enzyme activity by detecting the degree of decrease in NADH. NADH has a characteristic absorption peak at 340 nm. That is, the absorbance value at 340 nm changes with the change of NADH concentration. If the NADH concentration in the formula is higher, the initial value (absorbance value at 340 nm) is larger, the more NADH can be consumed by ACC enzyme, and the larger the maximum enzyme activity that can be detected; if the NADH concentration in the formula is lower, the initial value (absorbance value at 340 nm) is smaller, the less NADH can be consumed by 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 is also necessary to ensure that most samples consume enough NADH.

[0170] As shown in Table 7, when the NADH concentration is between 0.86 - 1.72 mg / mL, the ACC enzyme activity of the sample is the highest, and the difference is more significant for animal samples. When the NADH concentration is greater than 3.44 mg / mL, the ACC enzyme activity of the sample decreases because the excessive NADH concentration causes the absorbance value to exceed the instrument range, and the actual consumption of NADH cannot be detected; when the NADH concentration is less than 0.43 mg / mL, the ACC enzyme activity of the sample also shows a decreasing trend, indicating that the provided NADH concentration is too low, less than the amount of NADH required by the sample ACC enzyme, so the enzyme activity is low. Generally speaking, the optimal NADH concentration is between 0.86 - 1.72 mg / mL.

[0171] Example 8

[0172] To expand 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 influence of different concentrations of activators on the experimental results.

[0173] Step 1: Prepare relevant reagents according to Example 1 above.

[0174] Step 2: Prepare an extraction mixture according to the ratio of extraction solution 1:extraction solution 2 = 990 μL:10 μL according to the sample volume; after preparing a working solution according to the ratio of reagent 3:reagent 4:reagent 5:reagent 6 = 50 μL:50 μL:200 μL:200 μL, use it.

[0175] Step 3: Prepare reagent 1 with five preparation ratios of Mg 2+ :K + 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).

[0176] Step 4: Take 1 part of animal tissue and 1 part of plant tissue (animal tissue: mouse liver; plant tissue: sunflower leaf), use Reagent 1 with different ion concentration ratios, and measure and calculate the sample enzyme activity according to Steps 3, 4, and 5 in Example 3. The experimental results are shown in Table 8 below.

[0177] Table 8

[0178]

[0179] As shown in Table 8, when the Mg 2+ :K + concentration = 0.87:0 mg / mL, the sample ACC enzyme activity is normal; when the Mg 2+ :K + concentration = 0:1.6 mg / mL, the sample ACC enzyme activity decreases, and the difference is more significant for animal samples with higher enzyme activity. Therefore, adding Mg 2+ as an activator is crucial for the enzymatic reaction.

[0180] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A method for determining the enzyme activity of acetyl-CoA carboxylase, characterized in that: The detection reaction process is as follows: ； Specifically 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; 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.

2. The method according to claim 1, 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.

3. The method according to claim 2, 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.

4. The method according to claim 2, 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.

5. The method according to claim 4, 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.

6. The method according to claim 5, 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.

7. The method according to claim 6, 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.

8. The method according to claim 5, 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.

9. 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 1-3; the extraction mixture is as described in any one of claims 5-7.

10. Use of the method according to any one of claims 1 to 8 or the kit according to claim 9 in any of the following aspects: (1) In fatty acid synthesis, understand the fatty acid synthesis rate and oil content; (2) Understand the activity of fatty acid synthesis in plants; (3) 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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