阅读说明：本技术 一种新型的钠钾atp酶的检测方法 (Novel detection method of sodium-potassium ATP enzyme ) 是由 殷放宙 殷武 戴辉 李伟东 李林 于 2020-06-21 设计创作，主要内容包括：目前用于评估钠钾ATP酶活性较为常用的方法为放射性示踪法或比色法。但这两种方法均存在缺陷,前者需要使用放射性同位素,对实验环境要求高；后者则在检测准确度、灵敏度与重复性上相对较弱。本发明引入一种新方法进行细胞钠钾ATP酶活性的检测,通过电感耦合等离子体质谱法(ICP-MS)精确定量转运进细胞的铷离子,由此测得细胞内铷离子的转运率,进而评估钠钾ATP酶的活性。此方法具有无放射性、可靠、准确等优点。(The currently used method for evaluating the activity of sodium-potassium ATPase is a radioactive tracer method or a colorimetric method. However, both methods have defects, namely the former method needs to use radioactive isotopes, and has high requirements on experimental environment; the latter is relatively weak in detection accuracy, sensitivity and reproducibility. The invention introduces a new method to detect the activity of cell sodium potassium ATPase, and rubidium ions transferred into cells are accurately and quantitatively measured by inductively coupled plasma mass spectrometry (ICP-MS), so that the transfer rate of rubidium ions in cells is measured, and the activity of sodium potassium ATPase is further evaluated. The method has the advantages of no radioactivity, reliability, accuracy and the like.)

1. A novel detection method of sodium-potassium ATPase comprises the following steps:

(1) culturing cells according to a proper method, adding an RbCl solution, cracking, detecting protein, digesting, dispelling acid, and preparing a test solution.

(2) Rb single element standard solution is used as a standard substance, Yttrium Y single element standard solution is used as an internal standard substance, and the inductively coupled plasma mass spectrometry is adopted to detect Rb⁺The amount of intake of (1).

2. The method for detecting Na-K ATPase according to claim 1, wherein the cell culture and treatment method of step 1 is as follows: dissolving and diluting rubidium chloride by using Hank's solution containing calcium and magnesium; inoculating the normally cultured cells into a pore plate, directly adding a Hank's solution containing RbCl into a control group when the cells cover about 70-80% of the plate bottom, and adding a Hank's solution containing RbCl into an administration group after adding drug-treated cells; after culturing in an incubator, sucking out the Hank's solution containing RbCl, washing cells by using the Hank's solution without RbCl, cracking the cells, and determining the protein concentration by a bicinchoninic acid (BCA) method; adding concentrated nitric acid and internal standard Y to digest cell at room temperature, and adding H after digestion₂O₂The acid is removed until the brown gas disappears, and finally the solution is made to the appropriate volume with ultra pure water.

3. The method for detecting Na-K ATPase according to claim 1, wherein the amount of the cells cultured in normal state is adjusted to 4 × 10 in step 1⁵－5×10⁵seed/mL, inoculated into a well plate; when the cells cover about 70-80% of the plate bottom, 1mL of Hank's solution containing RbCl is added after the drug is added to treat the cells for 24 h; after 2h incubation in the incubator, the rbCl-containing Hank's solution was blotted and the cells were washed 3 times with RbCl-free Hank's solution; subsequently, the cells were lysed with lysis buffer, protein concentration was determined with BCA, additional 1mL of concentrated nitric acid and internal standard Y to a final concentration of 20. mu.g/L was added to digest the cells at room temperature for 2 h; after digestion was complete, 250 μ LH was added₂O₂The acid was removed at 100 ℃ for 2h until the brown gas disappeared, and finally the solution was made up to 40mL with ultra pure water.

4. The method for detecting Na-K ATPase according to claim 1, wherein step 2: using rubidium (Rb) single element standard solution. Method for preparing the sameIs prepared by using 2.5 percent of HNO₃Dissolving and diluting the aqueous solution (v/v) to a concentration of 12.5-1000 mug/L to be used as a standard solution; using yttrium (Y) single element standard solution and 2.5% HNO₃The internal standard mother liquor was prepared by dissolving and diluting the aqueous solution (v/v) to a concentration of 8 mg/L.

5. The method for detecting Na-K ATPase according to claim 1, wherein the parameters of step 2 inductively coupled plasma mass spectrometry are: radio frequency power, cooling gas flow, auxiliary gas flow, atomizer flow, sampling depth, peristaltic pump rotating speed, scanning mode, scanning times and repeated sampling times; the method comprises the following steps of 1100W of RF power, 15L/min of cooling air flow, 1.2L/min of auxiliary air flow, 0.99L/min of atomizer flow, 7.0mm of sampling depth, 24r/min of rotation speed of a peristaltic pump, single-point peak jump in a scanning mode, 20 times of scanning and 3 times of repeated sampling.

6. The method for detecting Na-K ATPase according to claim 1, wherein step 2 is characterized in that: the method for detecting the sample by adopting the inductively coupled plasma mass spectrometry comprises the step of calculating Rb according to a standard curve⁺The content is divided by the protein concentration to give Rb per gram of protein⁺Transport concentration as the final result.

Technical Field

The invention relates to the field of detecting ATPase activity, in particular to a method for detecting cell sodium potassium ATPase to rubidium ions (Rb) by an inductively coupled plasma mass spectrometry (ICP-MS)⁺) Transmembrane transport assay to assess the activity of sodium potassium ATPase.

Background

Sodium potassium ATPase (Na, K-ATPase), also known as Na⁺-K⁺The pump, discovered in 1957 by J.C.Skou, is composed of α and β subunits, α subunit is transmembrane protein with molecular weight of about 120kD, and has Na + and K + binding sites and ATPase activity, β subunitThe gene is a small subunit and is a glycoprotein with a molecular weight of about 50 kD. The Na-K ATPase exists in all eukaryotic cells, is a metal ion transporter, pumps out 3 Na ions and 2K ions in a reverse electrochemical gradient manner when consuming one ATP molecule, and keeps the uneven ion distribution of high K in the membrane and high Na outside the membrane, thereby maintaining the stability of the resting potential of the cell membrane, balancing ion, osmotic pressure and pH, and playing a very important role in cells. Scientific research shows that the sodium potassium ATP enzyme has very important function in normal metabolism of human body and has close relation with the occurrence of some diseases, such as cerebral edema, cataract, cystic fibrosis, epilepsy, migraine, hypertension and the like.

At present, the method for evaluating the activity of the sodium-potassium ATPase is mainly used for detecting the transport efficiency of the transport protein to radioactive 86Rb, is generally determined by a radioactive tracing method, and detects the 86Rb by adopting a gamma counter, but the method needs to use radioactive isotopes, has high requirements on experimental operating environment and limits the rapid detection of the sodium-potassium ATPase. The method also adopts a colorimetric method to detect the activity of sodium-potassium ATPase, and the principle is that the enzyme contains Na⁺，K⁺、Mg²⁺After incubation at 37 ℃ for 10min, ATP is decomposed into ADP and inorganic phosphorus (Pi) by the enzyme, ammonium molybdate in the phosphorus determination reagent reacts with phosphoric acid in the sample in the form of molybdic acid in an acidic environment to generate phosphomolybdic acid, the phosphomolybdic acid immediately converts to a blue reduction product, molybdenum blue, when a reducing agent exists, and the molybdenum blue has maximum absorption at the wavelength of 650-660 nm. The amount of newly produced Pi per mg of protein per hour was taken as the unit of activity of the enzyme, and the result was the total ATPase activity. Adding Na into buffer solution⁺，K⁺Ouabain, a specific inhibitor of ATP-ase, obtained as Mg²⁺-ATP activity, the difference between the two being Na⁺，K⁺-ATPase activity. The colorimetric method has low requirements on experimental environment, but has low sensitivity.

Disclosure of Invention

The purpose of the invention is as follows: provides an ICP-MS (Inductively coupled plasma spectrometry) detection method based on Inductively coupled plasma spectrometry, and a method for determining the activity of sodium potassium ATPase of a eukaryotic cell membrane.

The technical scheme is as follows: aiming at the situation in the background art, the invention selects⁸⁶Rb-chloride (RbCl), a nonradioactive substitute for Rb, was determined by Inductively coupled plasma mass spectrometry (ICP-MS)⁺The activity of sodium potassium ATPase was assessed by transmembrane transport. Rb⁺And K⁺Is homologous in the same way over Na⁺，K⁺-uptake of ATPase into cells, and Rb⁺Is ions which do not exist in cells under the natural state, has small environmental interference and is easy to detect. Thus, detection of Rb transported into cells by the instrument⁺The activity of sodium potassium ATPase can be estimated.

ICP-MS is an element analysis technology, and has high detection sensitivity and high-efficiency sample analysis capability. ICP-MS instruments use plasma (ICP) as the ion source and Mass Spectrometry (MS) analyzers detect the generated ions, which can measure most elements of the periodic table simultaneously, and current ICP-MS has been developed as the most sensitive and versatile tool for element-specific detection and quantification. The method utilizes ICP-MS to establish the detection of cell sodium potassium ATP enzyme pair Rb⁺The activity of the sodium-potassium ATPase is evaluated by the method of ion transmembrane transport amount, so that the danger of a radioisotope determination method is avoided, and the sensitivity of sample detection is ensured.

The technical scheme is as follows:

a novel detection method of sodium-potassium ATPase comprises the following steps:

1. a novel detection method of sodium-potassium ATPase comprises the following steps:

(1) culturing cells by proper method, adding RbCl solution, cracking, detecting protein, digesting, removing acid,

Preparing a test solution.

(2) Rb (Rb) single element standard solution is used as a standard substance, yttrium (Y) single element standard solution is used as an internal standard substance, and the inductively coupled plasma mass spectrometry (ICP-MS) is adopted to detect Rb⁺The amount of intake of (1).

Rubidium chloride (RbCl) is dissolved and diluted by Hank's solution containing calcium and magnesium. Seeding the well with normally cultured cellsIn the plate, and until the cells cover about 70-80% of the plate bottom, the control group was added directly to the rbCl-containing Hank's solution, and the administration group was added to the drug-treated cells and then to the rbCl-containing Hank's solution. After incubation in the incubator, the RbCl-containing Hank's solution was aspirated, the cells were washed with RbCl-free Hank's solution, lysed, and the protein concentration was determined by the bicinchoninic acid (BCA) method. Adding concentrated nitric acid and internal standard Y to digest cell at room temperature, and adding H after digestion₂O₂The acid is removed until the brown gas disappears, and finally the solution is made to the appropriate volume with ultra pure water.

The cells cultured normally were adjusted to 4 × 10⁵－5×10⁵one/mL, seeded into well plates. When the cells covered approximately 70-80% of the plate bottom, 1mL of Hank's solution containing RbCl was added 24h after drug-treated cells were added. After 2h incubation in the incubator, the rbCl-containing Hank's solution was blotted and the cells were washed 3 times with RbCl-free Hank's solution. Subsequently, the cells were lysed with lysis buffer, the protein concentration was determined with BCA, 1mL of concentrated nitric acid was added and the cells were digested at room temperature for 2h to reach a final concentration of 20. mu.g/L of internal standard Y. After digestion was complete, 250 μ LH was added₂O₂The acid was removed at 100 ℃ for 2h until the brown gas disappeared, and finally the solution was made up to 40mL with ultra pure water.

Using rubidium (Rb) single element standard solution and 2.5% HNO₃Dissolving and diluting the aqueous solution (v/v) to a concentration of 12.5-1000 mug/L to be used as a standard solution; using yttrium (Y) single element standard solution and 2.5% HNO₃The internal standard mother liquor was prepared by dissolving and diluting the aqueous solution (v/v) to a concentration of 8 mg/L.

Parameters of inductively coupled plasma mass spectrometry are: radio frequency power, cooling air flow, auxiliary air flow, atomizer flow, sampling depth, peristaltic pump rotating speed, scanning mode, scanning times and repeated sampling times.

The method comprises the following steps of 1100W of RF power, 15L/min of cooling air flow, 1.2L/min of auxiliary air flow, 0.99L/min of atomizer flow, 7.0mm of sampling depth, 24r/min of rotation speed of a peristaltic pump, single-point peak jump in a scanning mode, 20 times of scanning and 3 times of repeated sampling.

Detecting the sample by ICP-MS method, and calculating Rb according to the standard curve⁺The content is divided by the protein concentration to give Rb per gram of protein⁺Transport concentration as the final result.

Has the advantages that: the ICP-MS method introduced by the invention can be used for detecting the activity of sodium-potassium ATPase. Sodium potassium ATPase activity was assessed by ICP-MS measurement of radioactivity-free rubidium chloride (RbCl) and was verified on cells under inhibition with the sodium potassium ATPase inhibitor rhein. Compared with the original radioactive isotope tracing method with radioactive danger and the colorimetric method with low sensitivity, the method has the advantages of safety, stability, rapidness and reliability, and solves the technical problems of high requirement on experimental environment, low sensitivity and the like in the prior art.

Drawings

FIG. 1 is a chart showing the results of the colorimetric detection of rhein inhibiting the activity of sodium potassium ATPase in HCT116 cells. The left side shows the results of detection after 60, 80, 120. mu.M rhein and 50nM ouabain treatment for 24h, and the right side shows the results of detection after 80. mu.M rhein treatment for 2, 6, 12, 24 h.^*p<0.05,^NSp >0.05, compared to the blank control.

FIG. 2 is a diagram of the results of an independent experiment for detecting the activity of sodium-potassium ATPase by a colorimetric method. HCT-116 cells were treated with 80. mu.M rhein and 50nM ouabain for 24h and the cell activity was measured colorimetrically, 3 times independently under the same conditions.^*p<0.05，^NSp >0.05, compared to the blank control.

FIG. 3 is a graph of the Rb standard for ICP-MS to detect Na-K ATPase activity. The arrangement was 12.5, 25, 50, 100, 200, 500, 800, 1000. mu.g.L^-1And (3) drawing a standard curve after the rubidium ion standard solution, wherein y is 0.024x-0.7336, and r is 0.9990.

FIG. 4 is a graph showing the result of ICP-MS detecting the sodium potassium ATPase activity of HCT116 cells inhibited by rhein. HCT116 cells were treated with 60, 80, 120. mu.M rhein and 1. mu.M ouabain for 24h, and ICP-MS measured the amount of rubidium ions transported in the cells, divided by the protein concentration of each sample to give the final Na-K ATPase activity (n. 6).^*p<0.05，^**p<0.01，^NSp >0.05 compared to the blank control.

FIG. 5 is an independent experiment of ICP-MS for detecting rhein to inhibit the activity of HCT-116 cell Na-K ATPaseAnd (5) a result chart. HCT-116 cells were treated with 80. mu.M rhein for 24h, and the amount of rubidium ions transported in the cells was measured by ICP-MS, and the final sodium potassium ATPase activity was determined by dividing the protein concentration of each sample. The detection is carried out 3 times independently under the same conditions.^**p<0.01,^***p<0.001，^NSp >0.05, compared to the blank control.

Detailed Description

The following embodiments illustrate the present invention in detail, but do not limit the invention in any way.