METHIONINE ADENOSYLTRANSFERASE (MAT) BIOLOGICAL ACTIVITY ASSAY AND DETECTION KIT

Abstract

The present invention discloses a methionine adenosyltransferase (MAT) activity assay method and a kit for measuring MAT activity. A sample and relevant reagents are mixed in certain way that MAT-catalyzed reaction occurs efficiently. The reaction and the competitive ELISA that quantifies the product S-adenosylmethionine (SAM) are carried out simultaneously. The MAT activity is calculated as the amount of SAM produced per unit time. SAM is calculated through spectral absorbance of the SAM produced and comparing it to that of the standard. The method of SAM quantification is via tracer-labelled anti-SAM antibody or SAM (or SAM analog) antigen through competitive ELISA, so that the produced SAM competes with the SAM antigen for binding anti-SAM antibody. The method and the kit described in the present invention are more sensitive, accurate, reliable, straightforward, easier and faster. The method was used to measure the MAT activities of normal and cancerous liver cells.

Claims

1. The use of anti-S-adenosylmethionine-specific antibodies for determining the biological activity of methionine adenosyltransferase.

2. A method for determining the biological activity of methionine adenosyltransferase in a sample, said method comprising the following steps: (a) reacting in a buffer system that preserves the biological activity of methionine adenosyltransferase, said sample containing said enzyme and substrates to form S-adenosyl-methionine; and (b) using an immunological method to measure the concentration of S-adenosylmethionine to determine whether methionine adenosyltransferase activity is present in said reaction system and determine the level of said methionine adenosyltransferase activity.

3. The method according to claim 2, wherein the substrate contains a buffer system of methionine, adenosine triphosphate, magnesium ions and potassium ions with suitable acidity and alkalinity.

4. The method according to claim 2, wherein the sample is derived from the group consisting of: genetic engineering products, purified samples from biological tissue cells, methionine adenosyltransferases in tissue cells, biological fluids or tissue and cell cultured fluids; biological fluids including blood, plasma, serum, saliva, urine, cerebrospinal fluid, abdominal or thoracic exudates and tissue fluids.

5. The method according to claim 2, wherein said immunological method includes using one of the following components: (a) a medium coated with S-adenosylmethionine or its analogues, or a medium coated with an anti-S-adenosylmethionine specific antibody, (b) correspondingly, a tracer-labeled anti-S-adenosylmethionine specific antibody, or a tracer-labeled S-adenosylmethionine or its analogues, and (c) a corresponding tracer detection system.

6. The method according to claim 2, wherein the catalytic reaction of methionine adenosyltransferase and immunoassay to measure the resulting product S-adenosylmethionine that competes with the coated S-adenosylmethionine antigen for the binding of tracer-labeled anti-S-adenosylmethionine antibodies, or competes with tracer-labeled S-adenosylmethionine antigens for the binding of coated anti-S-adenosylmethionine antibodies, can be simultaneous in one reaction system in one-step format.

7. The method according to claim 2, wherein the catalytic reaction of methionine adenosyltransferase and immunoassay to measure the resulting product S-adenosylmethionine that competes with the coated S-adenosylmethionine antigen for the binding of tracer-labeled anti-S-adenosylmethionine antibodies, or competes with tracer-labeled S-adenosylmethionine antigens for the binding of coated anti-S-adenosylmethionine antibodies, can be simultaneous in one reaction system in two-step format.

8. An assay kit for determining the biological activity of methionine adenosyltransferase comprising the following components: (a) a micro-titer plate coated with S-adenosyl-methionine antigen or its analogues, or anti-S-adenosylmethionine antibody; (b) a tracer labeled anti-S-adenosylmethionine antibody or tracer labeled S-adenosyl-methionine antigen or labeled S-adenosylmethionine analog; (c) an S-adenosylmethionine standard; (d) an enzyme substrate comprising methionine, adenosine triphosphate in appropriate buffer; (e) an MAT positive control; (f) a tracer detection system; and (g) a buffer system with appropriate pH.

9. The assay kit according to claim 7, wherein the said micro-titer plate is coated with polylysine, bovine serum albumin or other carrier protein-conjugated S-adenosylmethionine or S-adenosylmethionine analogues, or it is coated directly with anti-S-adenosylmethionine antibodies (monoclonal antibodies or polyclonal antibodies) or indirectly with the S-adenosylmethionine monoclonal antibody through goat or rabbit anti-mouse IgG antibody.

10. A one-step method for determining the biological activity of methionine adenosyltransferase in a sample using the assay kit according to claim 8, said method comprising the following steps: (a) add different concentrations of the standard solutions from said kit to the wells of a micro-titer plate from said kit coated with protein-SAM (or SAM analogues) conjugates or a polymer-SAM (or SAM analogues) conjugates to create standard curves; (b) add samples to be tested and methionine adenosyltransferase positive control to other wells; (c) add horseradish peroxidase or alkaline phosphatase conjugated anti-S-adenosylmethionine monoclonal antibody, methionine and adenosine triphosphate substrate buffer, mix and incubate at 37? C. for 60 minutes and wash the plate after incubation; (d) add horseradish peroxidase or alkaline phosphatase substrate, allow color development at 37? C. for 15 minutes followed by stop solution to terminate the reaction and then in the plate reader read optical absorbance at 450 nm wavelength OD450 per well; (e) plot the graph according to the known concentrations of the standard and the corresponding values of OD450, the curve equation can be obtained; (f) the sample OD450 is then substituted into the equation to obtain the corresponding concentration of SAM produced; and (g) calculate the methionine adenosyltransferase activity of the sample containing methionine adenosyltransferase based on the concentration of the synthesized SAM per unit time.

11. A two-step method for determining the biological activity of methionine adenosyltransferase in a sample using the assay kit according to claim 8, said method comprising the following steps: (a) add samples to be tested and methionine adenosyltransferase positive control to the methionine and adenosine triphosphate substrate buffer in separate tubes and incubate at 37? C. for 20 minutes; (b) add the mixtures from step (a) and different concentrations of said standard solutions to the wells of a micro-titer plate from said kit coated with protein-SAM (or SAM analogues) conjugates or a polymer-SAM (or SAM analogues) conjugates to create standard curves; (c) add horseradish peroxidase or alkaline phosphatase conjugated anti-S-adenosylmethionine monoclonal antibody, mix and incubate at 37? C. for 40 minutes and wash the plate after incubation; (d) add horseradish peroxidase or alkaline phosphatase substrate, allow color development at 37? C. for 15 minutes followed by stop solution to terminate the reaction and then in the plate reader read optical absorbance at 450 nm wavelength OD450 per well; (e) plot the graph according to the known concentrations of the standard and the corresponding values of OD450, the curve equation can be obtained; (f) the sample OD450 is then substituted into the equation to obtain the corresponding concentration of SAM produced; and (g) calculate the methionine adenosyltransferase activity of the sample containing methionine adenosyltransferase based on the concentration of the synthesized SAM per unit time.

12. The method according to claim 5, wherein the tracer is selected from the group consisting of enzymes, fluorescein, colloidal gold, chemiluminescent substances, biotin, digoxin (or digoxigenin), radiolabeled substances and various types of latex microspheres.

13. The method according to claim 6, wherein the tracer is selected from the group consisting of enzymes, fluorescein, colloidal gold, chemiluminescent substances, biotin, digoxin (or digoxigenin), radiolabeled substances and various types of latex microspheres.

14. The method according to claim 7, wherein the tracer is selected from the group consisting of enzymes, fluorescein, colloidal gold, chemiluminescent substances, biotin, digoxin (or digoxigenin), radiolabeled substances and various types of latex microspheres.

15. The assay kit according to claim 8, wherein the tracer is selected from the group consisting of enzymes, fluorescein, colloidal gold, chemiluminescent substances, biotin, digoxin (or digoxigenin), radiolabeled substances and various types of latex microspheres.

16. The assay kit according to claim 8, wherein the buffer system comprises a magnesium ion concentration in the range of about 20-100 mM, a potassium ion concentration in the range of about 50-400 mM, a tris (hydroxymethyl) aminomethane hydrochloric acid concentration in the range of about 50-200 mM, and a pH range of about 7.42-8.5.

17. The assay kit according to claim 8, wherein said buffer system comprises: (a) a tris (hydroxymethyl) aminomethane HCl buffer having a concentration of about 100 mM to about 200 mM to provide a pH from about 7.42 to about 8.5; (b) a magnesium salt suitable to provide a magnesium ion concentration in the range of about 50 mM to about 100 mM; and (c) a potassium salt to provide a potassium ion concentration in the range of about 50 mM to about 200 mM.

Description

DESCRIPTION OF THE DRAWINGS

[0050] In order to clearly describe the technical solutions in the embodiments of the present invention or in the prior art, the drawings used in the description of the embodiments or the prior art are briefly described below.

[0051] FIG. 1 is a schematic of a direct competitive ELISA steps and components. The figure shows an example of an antigen instead of an antibody bound to the plate.

[0052] FIG. 2 illustrates the effects of methionine adenosyltransferase concentration, amount of methionine substrates, and pH of the reaction buffer on the synthesis of S-adenosyl-methionine. The synthesis yield of SAM was expressed as A/A0, i.e., the ratio of sample well OD450 to control well OD450; the lower the A/A0, the greater the ability of the samples or standards to compete with the coating antigen for HRP-labeled antibody. The higher the SAM production, the greater is the MAT activity.

[0053] FIG. 3 shows the determination of the optimal reaction time in the direct determination of MAT activity in vitro. MAT enzyme catalyzed the substrates to generate SAM in a straight rising phase before 60 min; then within 60-90 min, the SAM content did not change significantly and was in the plateau phase, indicating that the concentration of substrates might be insufficient or the enzyme activity might be lost after 1 h, leading to no more SAM production. The reaction was faster when the enzyme concentration was 1.0 mg/ml rather than 0.6 mg/ml.

[0054] FIG. 4 describes the regulation of MAT activities by nitrosoglutathione (GSNO) and Met in normal liver cell line L02 and liver cancer cell line HepG2. The ordinate is the SAM concentration by MAT catalyzed synthesis. The activities of MAT-I/M in L02 cells and MAT-II in HepG2 cells were regulated by GSNO and different concentrations of Met. For L02 cells, 500 ?M Met stimulated MAT-III/I activity, 2 mM Met stimulation was not significant. The inhibitory effect of GSNO nitrosylation on MAT-III/I was obvious. In HepG2 cells, Met inhibits MAT-II activity, and GSNO has no inhibitory effect on MAT-II.

[0055] FIG. 5A to FIG. 5D show a comparison of the results of immunofluorescence staining using anti-SAM monoclonal antibody at 1:400 dilution before and after stimulation with 0.5 mM Met for 24 h on normal hepatocyte L02 and hepatocellular carcinoma HepG2 (?200). FIG. 5A features the results for L02, 0 nM Met; while FIG. 5B describes the results for L02, 0.5 mM Met 24 h. FIG. 5C shows the results for HepG2, 0 nM Met; while FIG. 5D shows results for HepG2, 0.5 mM Met 24 h. On a black background, green fluorescent (light-colored) positive cells showed SAM expression, and black background showed no or very low SAM. It can be seen that Met stimulated MAT-III/I activity of L02 cells, and thus SAM expression increased in group shown in FIG. 5B compared to group shown in FIG. 5A; conversely, Met inhibited the activity of MAT-II in HepG2 cells, and thus SAM expression in group of FIG. 5D was lower than in the FIG. 5C group.

DETAILED EXAMPLES OF THE INVENTION

[0056] The following further describes the present invention in detail with reference to the experimental results and data. Raw materials not mentioned are conventional commercial reagents and are commercially available.

Example 1

[0057] Cross-Reactivity of SAM and Methionine Adenosyltransferase Substrates ATP and L-Met Experimental Materials

[0058] Anti-SAM and HRP-conjugated anti-SAM antibody: Arthus Biosystems, MA00201, MA00202, PA00201, MAH00201; polylysine (PLL) or bovine serum albumin (BSA) conjugated SAM antigens (for coating ELISA plates): Arthus Biosystems, ACT00201, ACT00204; S-adenosylmethionine (aza-SAM) standard: Arthus Biosystems, AST00201; S-adenosylmethionine (adenosine methionine disulfide methionine, SAMe) standards: Sigma, A2408, and the same named purchased from Shaanxi Pioneer Biotechnology Co., Ltd.; Methionine adenosyltransferase (MAT): Beijing Aibixin Biotech Co., Ltd. Abt-P-005; BSA, Tris, also known as tris (hydroxymethyl) aminomethane, NaCl, NaH.sub.2PO.sub.4, Na.sub.2HPO.sub.4: Beijing Huameijiacheng Biotechnology Co., Ltd.; KCl: Tianjin Damao Chemical Reagents Co. Ltd.; MgSO.sub.4: Hunan Honghao Gene Technology Co., Ltd.; Adenosine triphosphate (ATP): Shanghai Jingchun Chemical Reagents Co., Ltd.; L-Met, ProClin 300: Sigma; TMB: Huzhou Yingchuang Biotechnology Co., Ltd.; Sulfuric acid: Hunan Kangdu Pharmaceutical Co., Ltd.; 96-well enzyme-linked immunoassay micro-titer plate: US Corning high adsorption enzyme-linked immunoassay plate.

Reagent Preparation

[0059] Enzyme Reaction Buffer: 100 mM Tris, 100 mM KCl, 20 mM MgSO.sub.4, ProClin 1%, adjusted pH values pH 7.42, pH 8.0, pH 8.5 respectively; SAM analogue standard (aza-SAM): 960 nM, 480 nM, 240 nM, 120 nM, 60 nM, 30 nM, 15 nM, 0 nM, in enzyme reaction buffer pH7.42; SAM analogous quality control: 200 nM aza-SAM in enzyme reaction buffer pH 7.42; SAM standard (SAMe): 1-40 ?M in enzyme reaction buffer pH7.42; SAM quality control in: 8 ?M SAMe in enzyme reaction buffer pH7.42; the enzyme reaction buffer: 0.5% or 0.2% IB (Incubation Buffer): 10 nM PB (Phosphate Buffer), 150 mM NaCl, 0.5% or 0.2% BSA, 0.1% ProClin.

[0060] The ELISA plates were coated with PLL SAM antigen. The SAM standard curve was created using 0, 15, 30, 60, 120, 240, and 480 nM standards IB containing 0.5% BSA. The concentrations of the cross-reacting substances ATP and L-Met were 0, 120, 1200, 3960 and 12000 nM. The HRP-anti-SAM antibody was diluted at 1:25000 with HRP antibody diluent. Added 30 ?l standards and cross-reacting substances ATP and Met per well, and 70 ?l diluted HRP-anti-SAM antibody per well and incubated at 37? C. for 1 h. Washed the plate, added 100 ?l TMB at 37? C. for 15 min, added 50 ?l stop solution and read OD450. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Reaction rates of anti-SAM monoclonal antibodies with ADP and Met (A/A0) SAM nM SAM standard ATP L-Met nM Cross-reaction (%) 0 1 1 1 0 15 0.793999439 1.0513698 1.0421467 120 50% 30 0.723637723 1.0007116 1.0464435 1200 10% 60 0.585606131 1.094967 1.0118777 3960 3% 120 0.430096271 1.0861819 1.0953228 12000 1% 240 0.271109449 480 0.123357323 Cross- <1% <1% reaction (%)
A/A0; reaction rate (A0 is the OD450 reading at 0 nM, A represents any OD450 reading of wells that have different amount of free small molecule antigen competing.)

[0061] Cross-reaction rate=(Concentration of free antigen when 50% inhibition is achieved/Concentration of cross-reaction substances when 50% inhibition is achieved)*100%. The results of repeated experiments showed that the cross-reaction rates of ATP, ADP and Met with anti-SAM monoclonal antibodies 118-6, 84-3 and rabbit anti-SAM polyclonal antibody. R3 were much less than 1%. The cross-reaction of anti-SAM antibody with methionine, adenosine, S-adenosylhomocysteine and methylthioadenosine are all far less than 1% (see antibody product data from Arthus Biosystems). Therefore, this experiment does not have any cross-reaction with other components in the reaction system.

Example 2

Enzyme Activity Assay System: Optimum Reaction Ratio of Methionine Adenosyltransferase, Methionine and Adenosine Triphosphate Under Different pH Conditions

[0062] Using ELISA strips that were pre-coated with PLL-SAM antigen, and different concentrations of ATP, Met, and MAT enzyme were formulated with the enzyme reaction buffer at pH 7.42, pH 8.0, and pH 8.5 respectively. As shown below, codes A\B\C\D represents four Met concentrations, adenosine triphosphate components, and code 1\2\3 represents three MAT concentrations. The combination of the two codes was used to prepare the chessboard design. A total of 12 different combinations of methionine adenosyltransferase, methionine, adenosine triphosphate solutions have been obtained.

TABLE-US-00002 Code MAT 1 0.3 mg/ml 2 0.6 mg/ml 3 1 mg/ml

TABLE-US-00003 Code ATP Met A 2.4 mM 0.2 mM B 2.4 mM 0.8 mM C 2.4 mM 1.6 mM D 2.4 mM 2.4 mM

[0063] The reaction was performed at 37? C. for 1 hour. After the plate was washed, 100 ?L of TMB was added to each well. After incubation at 37? C. for 15 minutes, 50 ?l of the stop solution was added before reading OD450. The results are shown in Table 2 below.

TABLE-US-00004 TABLE 2 Comparison of OD450 values from MAT-catalyzed synthesis reactions under different substrate concentrations, MAT levels and pH conditions OD450 design pH = 7.42 1.9715 1.8155 1.0839 1.0683 A1 A1 A3 A3 1.6422 1.4980 0.8157 0.7668 B1 B1 B3 B3 1.4120 1.3400 0.7347 0.7082 C1 C1 C3 C3 1.3852 1.2713 0.6608 0.6588 D1 D1 D3 D3 1.2270 1.1315 2.6735 2.9059 A2 A2 A 0 competition background pH = 7.42 1.0224 0.9309 2.6567 2.7983 B2 B2 B 0 competition background pH = 7.42 0.9868 0.8753 2.7033 0.0488 C2 C2 C blank background 0.9889 0.9092 2.8659 0.0470 D2 D2 D blank background pH = 8.0 2.1943 2.1483 1.1701 1.2561 A1 A1 A3 A3 1.8445 1.9023 0.8043 0.8439 B1 B1 B3 B3 1.7334 1.7538 0.7860 0.8290 C1 C1 C3 C3 1.7352 1.8867 0.7320 0.7191 D1 D1 D3 D3 1.4195 1.3596 2.4978 2.7377 A2 A2 A 0 competition background pH = 8.0 1.1351 1.0597 2.5387 2.7492 B2 B2 B 0 competition background pH = 8.0 0.9960 0.9884 2.5973 0.0487 C2 C2 C blank background 1.0172 1.0253 2.7131 0.0461 D2 D2 D blank background pH = 8.5 1.6684 1.9050 1.0299 1.0203 A1 A1 A3 A3 1.2567 1.5798 0.6783 0.7050 B1 B1 B3 B3 1.1811 1.3139 0.5387 0.5755 C1 C1 C3 C3 1.1386 1.2621 0.5609 0.5186 D1 D1 D3 D3 0.8358 0.8323 2.4289 2.7261 A2 A2 A 0 competition background pH = 8.5 0.6387 0.8024 2.5742 2.7016 B2 B2 B 0 competition pH = background 8.5 0.6104 0.6657 2.5285 0.0468 C2 C2 C blank background 0.6390 0.7459 2.6965 0.0484 D2 D2 D blank background

[0064] The synthetic yield of SAM is expressed as A/A0, i.e. the ratio of OD450 of sample well to control well. The lower the A/A0, the greater the ability of the SAM synthesized to compete with the coating antigen for HRP-labeled antibodies. The higher the SAM production, the greater is the MAT activity. The results are shown in Table 3 below.

TABLE-US-00005 TABLE 3 Comparison of relative amounts of SAM catalyzed by MAT under different substrate concentrations, MAT levels and pH conditions MTA 0.3 MTA 0.6 MTA 1.0 A/A0 pH = 7.42 A1 0.658156 A2 0.403448 A3 0.366664 B1 0.542829 B2 0.3312 B3 0.265085 C1 0.473611 C2 0.314938 C3 0.240193 D1 0.456583 D2 0.321357 D3 0.218208 A/A0 pH = 8.0 A1 0.787768 A2 0.497812 A3 0.432365 B1 0.677275 B2 0.389451 B3 0.288083 C1 0.629131 C2 0.350432 C3 0.281926 D1 0.654111 D2 0.361207 D3 0.25153 A/A0 pH = 8.5 A1 0.652252 A2 0.294959 A3 0.366613 B1 0.514064 B2 0.252391 B3 0.241552 C1 0.450024 C2 0.221449 C3 0.191089 D1 0.432341 D2 0.241852 D3 0.184582

[0065] The resulting reaction rate, i.e. the A/A0 and methionine concentrations were plotted to obtain the results shown in FIG. 2. (1) At different pH conditions, when the amounts of substrate and enzyme are constant, the A/A0 at pH 8.5 is minimized and is the highest at pH 8.0. However, the production of SAM increase was not very significant; (2) when the substrate ATP was unchanged (2.4 mM), Met increased from 0.2, 0.8, 1.6, to 2.4 mM, the SAM production increased significantly; (3) When the amount of MAT enzyme increased, its SAM production increased rapidly.

[0066] Based on the results from three different conditions above, the strongest factor influencing the SAM formation reaction is the amount of MAT, followed by the increase in substrate Met, and the smallest factor is the buffer pH value. In addition, because the MAT used in this experiment is a product of E. coli MAT gene expression, it's catalytic activity might not be ideal, therefore the required substrate concentration was relatively higher, but the experiment shows that even in the milli-molar level of Met and ATP, there was no cross-reaction. Since the aim of this experiment was not to screen for highly active MAT, the MAT selected here is able to meet the needs of establishing a MAT activity determination methods. Due to the relatively high activity of MAT under alkaline conditions such as pH 8.5, the antigen-antibody binding is reduced at pH 8.5 compared to pH 7.42, thus A/A0 from FIG. 2 appeared relatively low at pH 7.42 and pH 8.5 (i.e, the amount of SAM synthesis was high), and the A/A0 was relatively high at pH 8.0 (i.e, the amount of SAM synthesis was small). Therefore, the best MAT measurement system should take into account various factors. The pH value is set to be either 7.42 or 8.5.

Example 3

Determination of the Optimal Reaction Time for Measurement of MAT Activity In Vitro

[0067] Take PLL-SAM-coated antigen ELSIA strips, and add SAM standards at concentration of 0, 15, 30, 60, 120, 240, 480, 960 nM, and quality control 200 nM that were prepared in an enzyme reaction buffer at pH 7.42. Add ATP and Met at 2.4 mM, plus MAT enzyme at 0.6 mg/ml, or ATP and Met at 2.4 mM, plus MAT at 1.0 mg/ml (all final concentrations) as SAM substrate and enzyme mixture. Add 30 ?l per standard well and enzyme samples, and 70 ?l per well of HRP-anti-SAM antibody. Then incubated at 37? C. for 30 min, 60 min, 90 min, after washing the plate, 100 ?l TMB was added and left at 37? C. for 15 min, added 50 ?l stop solution and read OD450. Results are shown in Table 4.

TABLE-US-00006 TABLE 4 Effect of different reaction times on the amount of SAM catalyzed by MAT Concentration (nM) 30 min 60 min 90 min ATP and Met 2.4 mM, MAT 116.3030073 221.5053 204.34158 0.6 mg/ml ATP and Met2.4 mM, MAT 270.8731347 396.8892 410.82783 1.0 mg/ml

[0068] The results above indicated that the amount of SAM synthesized at 60 minutes was significantly higher than that at 30 minutes, but was almost the same at 90 minutes and 60 minutes. Measured SAM synthesis from the 20-90 minutes time period, the relationship of SAM production change over time could be obtained.

[0069] Take PLL-SAM-coated antigen ELSIA strips, and add SAM standards and quality control standards were prepared in an enzyme reaction buffer at pH 7.42, added ATP and Met at 2.4 mM, plus MAT enzyme at 0.6 mg/ml, or ATP and Met at 2.4 mM, plus MAT at 1.0 mg/ml (all final concentrations) as SAM substrate and enzyme mixture. Added 30 ?l per standard well and enzyme samples, and 70 ?l per well of HRP-anti-SAM antibody. Incubation time at 37? C. is shown in Table 5. After washing the plate, 100 ?l TMB was added and left at 37? C. for 15 min, added 50 ?l stop solution and read at OD450. Data processing is the same as done previously. The concentrations measured are as follows.

TABLE-US-00007 TABLE 5 Relationship between reaction time and SAM synthesis Reaction SAM yield (nM) over time Conditions 20 min 30 min 40 min 50 min 60 min 70 min 80 min ATP and Met 124.28 158.98 194.17 215.13 255.39 268.26 264.06 (2.4 mM), MAT (0.6 mg/ml) ATP and Met 220.63 274.60 335.53 374.90 433.83 445.82 450.21 (2.4 mM), MAT (1.0 mg/ml) The corresponding MAT activity (U/mg) ATP and Met 10.35667 8.832095 8.090302 5.837829 7.094171 6.38704 5.188694 (2.4 mM), MAT (0.6 mg/ml) ATP and Met 11.03143 9.153337 8.388306 7.298031 7.647138 6.225992 5.627597 (2.4 mM), MAT (1.0 mg/ml)

[0070] The concentration of synthesized SAM and reaction time were plotted and the results are shown in FIG. 3. The experimental results have been verified several times under different buffer systems and different MAT conditions. The results showed that the amount of SAM synthesized by MAT was in a straight line rising phase before 60 min, and the amount of SAM did not change significantly during the period of 60-80 min, i.e. in a plateau phase, indicating that the concentration of substrate might not be enough or the enzyme activity was lost after 1 h, resulting in no increase in SAM production. The reaction rate was faster when the enzyme concentration was 1.0 mg/ml rather than 0.6 mg/ml.

Example 4

The Optimal Buffer System for Determination of MAT Activity In Vitro

[0071] Using a system similar to the example above, the coating PLL-aza-SAM was used at 0.05 ?g/ml. The HRP-anti-SAM antibody 118-6 was diluted at 1:30000 and was incubated for 1 hour. The pH value of the prepared solution was 7.40?0.05, and the ATP was at 5 mM, Met at 4 mM, MAT at 1 mg/ml. When titrating the concentration of Mg.sup.2+, the buffer contained 250 mM KCl and 100 mM Tris. When titrating the concentration of K.sup.+, the buffer contained 20 mM MgSO.sub.4 and 100 mM Tris. After the optimal concentrations of Mg.sup.2+ and K.sup.+ were determined, Tris was titrated again. The results are shown in Table 6. The A/A0 was gradually decreased by 0.475 when the concentration of MgSO.sub.4 was from 4 mM to 100 mM. However, A/A0 decreased rapidly when the concentration of MgSO.sub.4 was from 4 to 50 mM, and decreased slowly when the concentration of MgSO.sub.4 was from 50 to 100 mM. Thus, the suitable MgSO.sub.4 concentration was selected between 50 mM and 100 mM. The A/A0 has small changes when the concentration of KCl was from 50-400 mM, There was no significant decrease and increase trend, and chose about 150 mM. The concentration of Tris was 100 mM and remained optimal.

TABLE-US-00008 TABLE 6 Relationship between reaction time and SAM synthesis MgSO.sub.4 KCl Tris Conc. (mM) A/A0 Conc. (mM) A/A0 Conc. (mM) A/A0 4 0.2199 50 0.1983 20 0.4981 10 0.2047 100 0.1938 50 0.2567 30 0.1867 150 0.1911 100 0.2168 50 0.1766 200 0.1973 200 0.2472 70 0.1734 250 0.2079 100 0.1724 300 0.2106 400 0.1995

Example 5

Determine the Enzymatic Activity of MAT Purified from Mouse Liver Cells Under Optimal Reaction Conditions

Experimental Materials

[0072] Sterile PBS, 0.25% trypsin (Aladdin), 1640 medium (Gibco), 10% FBS (Yuanheng St. Ma), serum-free MEM medium (Gibco), 10 g/ml Met aseptic concentrate, GSNO, 15 cm.sup.2 square bottle, 75 cm.sup.2 square bottle (Corning), FITC-goat anti-mouse IgG (abcam), DAPI (Life Technologies); Triphenylene blue powder (Aladdin).

[0073] Phosphate buffer (PBS): NaCl 8 g, Na.sub.2HPO.sub.4..sub.12H.sub.2O 2.885 g, KCl 0.2 g, KH.sub.2PO.sub.4 0.2 g, ultrapure water 1000 ml; trypsin (0.25% trypsin): trypsin 0.1 g, 4% EDTA solution 200 ?l 39.8 ml of 1?PBS solution; Trypsin is fully dissolved and filtered with a 0.2 ?m sterile filter (Pall). Trypan blue solution (4% Triphenyl blue solution): Triphenyl blue 1.6 g, ultrapure water 40 ml, filtered with filter paper.

The experimental design is shown in Table 7.

TABLE-US-00009 TABLE 7 Experimental group parameters of GSNO pretreatment and Met on the intracellular MAT activity in L02 and HepG2 cells Number 1 2 3 4 5 GSNO 0 mM 0 mM 0 mM 1 mM 1 mM Met 0 mM 0.5 mM 2 mM 0.5 mM 2 mM

Experimental Steps:

[0074] 1. When the cells in the flask are at about 80% confluence, remove the 1640 medium (with 10% fetal bovine serum) and wash with PBS once;

[0075] 2. Take the two groups containing GSNO, treat the cells with serum-free MEM medium containing 1 mM GSNO and leave in the incubator for 30 min.

[0076] 3. Remove the serum-free medium containing GSNO and wash it with PBS once;

[0077] 4. Add Met-containing serum-free MEM medium (containing 5% fetal bovine serum and 2 mM glutamine) into the square flask according to the parameters in the table above. Add the Met-free MEM as the control group and put into the incubator for 24 h;

[0078] 5. Remove the culture medium from the flask and add an appropriate volume of trypsin to ensure that the pancreatin covers the bottom of the entire square flask;

[0079] 6. Place the flask in a 37? C., 5% CO.sub.2 incubator for 2-3 minutes. When cells start to fall out, add 5 ml of 1640 medium containing 10% FBS to terminate the action of pancreatin.

[0080] 7. Resuspend with about 1 ml of PBS after centrifugation, and count with trypan blue staining.

[0081] 8. Take 1 ml of each cell suspension, place on an ice bath and sonicate it (see Example 5). Centrifuge at 15000 g and then collect the supernatant.

[0082] 9. Reaction is performed as described in Example 3 at 37? C. for 60 min to determine the MAT activity in the cell supernatant.

[0083] Since the amount of cells in each experimental group used for the ELISA assay was not exactly the same, L02, HepG2 cells were normalized by cell count (adjusted to 2?10.sup.7). The amounts of SAM synthesized by MAT under different conditions are shown in Table 8 and FIG. 4.

TABLE-US-00010 TABLE 8 Effects of GSNO and Met on different MAT activities in the L02 and HepG2 cells HepG2 SAM (nM) L02 SAM (nM) 0 mM Met 247.4761 0 mM Met 274.4578162 0.5 mM Met 222.3993 0.5 mM Met 377.700459 2 mM Met 174.3621 2 mM Met 236.4367472 0.5 mM Met + 1 mM 200.6807 0.5 mM Met + 1 mM 226.6779748 GSNO GSNO 2 mM Met + 1 mM 198.1654 2 mM Met + 1 mM 176.6032683 GSNO GSNO

[0084] Human normal liver cell line L02 expresses the dimer MAT-III or tetramer MAT-I consisting of the catalytic subunit ?1 encoded by the MAT1a gene, whereas HepG2 hepatoma cells express tetramer MAT-II consisting of the catalytic subunit ?2 and regulatory subunit ? encoded by the MAT2a/2b gene. The responses to Met stimulation, cell types of expression and effects of the two MAT enzymes encoded by these two genes are not the same. The presence of MAT-III or MAT-I in adult hepatocytes is sensitive to Met stimulation, and its function is to rapidly reduce Met levels in the blood during high Met diets. Therefore, MAT-III or MAT-I activity is increased by Met stimulation [5]. Our results show that 500 ?M Met stimulated the activity of MAT-III or MAT-I in vitro for 24 hours, and the inhibition of MAT-III or MAT-I after nitrosation of GSNO was obvious. No stimulatory effects of Met at 2 mM dosage on MAT-III or MAT-I activity was observed, and MAT activity on the contrary was slightly decreased. The reason was unclear. However, GSNO nitrosylation-inhibitory effects on MAT activity in the presence of 2 mM Met remained obvious. In contrast, the dose-response relationship of Met inhibits MAT-II activity in hepatoma cell lines is consistent with the literature [12], i.e. the higher the Met concentration, the lower the activity of MAT-II. In addition, our results also showed that nitrosylation has a minor effect on MAT-II, which is in agreement with the reported elsewhere that NO in GSNO inhibits MAT activity by binding to the cysteine at position 121 of MAT-II [5]. GSNO mainly inhibits the activity of MAT-III/I post Met stimulation.

[0085] Immunofluorescence staining of these two cell lines was performed simultaneously with the competitive ELISA described above for quantification of SAM, the procedure is as follows:

[0086] (1) Digest the HepG2 and L02 cells from the bottom of the square flask;

[0087] (2) Centrifuge at 1050 rpm for 5 min and re-suspend the cells in 1640 medium containing 10% FBS in (Gibco);

[0088] (3) After counting with trypan blue, cells were seeded onto 24 wells at a density of 7.5?10.sup.4 cells/well, and 8 wells were seeded each cell line;

[0089] (4) Add the medium to 24-well culture plate to 1 ml/well and place it in the incubator for 24 h;

[0090] (5) Remove the original culture medium from the wells. Add 1 ml/well MEM medium containing 5% FBS and 500 ?M Met according to Table 1, and put the culture plate into the incubator for 24 h.

[0091] (6) After 24 h. discard the medium and wash it twice with 37? C. pre-warmed PBS. Add 500 ?l 80% ice acetone per well at ?20? C. for 20 min. Wash 3 times with PBS.

[0092] (7) Add primary antibody: Anti-SAM monoclonal antibody was diluted at 1:400 with PBS containing 0.5% skimmed milk, 50 ?L per well, 37? C. wet-box for 1 h, and PBS washed 3 times.

[0093] (8) Add secondary antibody: FITC-labeled goat anti-mouse IgG diluted at 1:500 with PBS containing 0.5% non-fat dry milk, 50 ?L per well, 37? C. wet-box for 45 min, PBS washed 5 times (to avoid light); observed by ordinary fluorescence microscope and took pictures.

[0094] (9) 100 ?L of 0.5 mg/ml DAPI (diamidino-2-phenylindole) was added and stained the cell nucleus for 20 min; after washing with PBS for 5 times, observed the cells under laser scan confocal microscope directly and photographed as needed.

[0095] FIGS. 5A through 5D show the comparison of the results of immunofluorescence staining of L02 and HepG2 cells after stimulation with 0.5 mM Met for 24 h and without Met under similar conditions as described above. Qualitative results indicated that SAM synthesis increased in L02 cells after 0.5 mM Met stimulation for 24 h, which was due to the increase of MAT-III/I activity; whereas the amount of SAM synthesis in Met treated HepG2 decreased since Met inhibited MAT-II activity. The results of immunofluorescence staining by laser scan confocal microscopy further confirmed the results above.

Example 6

Determination of MAT Activity Purified from Mouse Liver

Laboratory Equipment:

[0096] PPS protein purification system (Institute of Engineering, Chinese Academy of Sciences); Ultrasonic crusher (Ningbo Xinzhi); High-speed refrigerated centrifuge (Xiangli Centrifuge)

Reagent Preparation:

[0097] Tissue homogenates: sucrose 0.25 M, Tris 10 mM, EGTA 0.1 M, beta-mercaptoethanol 0.1%. pH 7.5; Buffer A: MgSO.sub.4 10 mM, EDTA 1 mM, Tris 10 mM, pH 7.5; Buffer B: Buffer A+600 mM KCl; Dialysate: Buffer A+75 mM KCl; pH7.42 Enzyme Reaction Buffer: 100 mM Tris, 100 mM KCl, 20 mM MgSO.sub.4, ProClin 1% adjusted to pH 7.42; 50% DMSO solution: Buffer A+equal volume of DMSO.

Liver Tissue Homogenate:

[0098] 1. Dissect mouse, remove connective tissue from liver, place it in a de-contracted 1.5 ml tube, and weigh the liver. The net weight was 0.6 g.

[0099] 2. Wash the liver 3 to 5 times with 1 ml normal saline until the solution is clear and transparent.

[0100] 3. The liver was cut into pieces and placed into a clean tissue homogenizer. About 3-fold more ice-cold tissue homogenate solution was added to the liver tissue pieces. Rotated and grinded in an ice bath for 6-8 min and grinded it thoroughly. The homogenate was removed, and the homogenizer was filled with 1 volume of hepatic tissue homogenate to wash the wall and aspirated into the homogenate.

[0101] 4. The homogenate was placed in the liquid ice bath in an ultrasonic crusher for comminution, with ?6 probe, power 80%, working time was 3 seconds, pause time was 3 seconds, sonication was 30 minutes.

[0102] 5. Homogenate was centrifuged under refrigerated condition at 15,000?g high speed for 20 min, the supernatant was filtered with qualitative filter paper. A total of 150 ml homogenate was obtained.

[0103] 6. Slowly added 31.3 g of ammonium sulfate per 100 ml of the homogenate with stir, and added 47 g of ammonium sulfate to make the saturation to be 50%. Stirred for 30 min and let it stand for 60 min.

[0104] 7. Supernatant from high-speed refrigerated centrifuge was discarded. The precipitate was dissolved with 150 ml of pre-cooled dialysate, dialyzed in 2 L dialysate, and exchanged once.

Purify with DEAE Column:

[0105] 1. Dialysis sample processing: The dialysis sample was taken out and centrifuged at high speed to discard precipitate. The supernatant is filtered with filter paper. About 230 ml of sample was obtained.

[0106] 2. Took 60 ml of DEAE packing, packed the column (3?20 cm), and applied to the PPS chromatography system, rinsed with deionized water at a flow rate of 10 ml/min for 30 min, and equilibrated with dialysis buffer.

[0107] 3. Mixed the sample and DEAE packing in a 4? C. refrigerator using a low speed shaker for 90 minutes.

[0108] 4. Packed the column, rinsed on the chromatographic system, and dialysis buffer was set to 6 ml/min for 30 min, set UV to zero.

[0109] 5. Elution: 100% buffer B salt eluted for 20 min with a linear gradient, flow-rate was 6 ml/min. When the UV monitoring peak appeared, started to collect 5 ml per tube till the end of the peak, A total of 38 tubes were collected.

MAT Activity Test for DEAE Eluent:

[0110] Used the PLL-SAM antigen coated ELSIA plate. Prepared MAT reaction system with reaction buffer with pH 7.42, ATP and Met at 2 mM, and elution sample accounted for 50% of the reaction mixture. Control group had not ATP and Met substrates. The SAM quantification kit was used to measure the amount of SAM produced by each eluent, which indirectly reflected MAT activity and elution range. The activity unit was defined as the amount of SAM in nM produced per minute per milligram of MAT enzyme.

TABLE-US-00011 TABLE 9 Reaction rates A/A0 of different DEAE eluent Elution 1 0.994 Elution 9 0.938 Elution 0.774 Elution 0.985 Elution 33 0.966 MAT 17 25 Elution 2 0.981 Elution 0.897 Elution 0.782 Elution 0.956 Elution 34 1.004 MAT 10 18 26 Elution 3 0.958 Elution 0.880 Elution 0.830 Elution 0.950 Elution 35 0.986 ATP + 11 19 27 Met Elution 4 0.954 Elution 0.829 Elution 0.843 Elution 0.975 Elution 36 0.951 Buffer 12 20 28 Elution 5 0.976 Elution 0.864 Elution 0.844 Elution 0.966 Elution 37 0.963 MAT 13 21 29 Control Elution 6 0.985 Elution 0.799 Elution 0.865 Elution 0.968 Elution 38 1.002 MAT 14 22 30 Control Elution 7 0.971 Elution 0.732 Elution 0.878 Elution 0.995 Pre- 0.956 Blank 15 23 31 elution Elution 8 0.961 Elution 0.688 Elution 0.949 Elution 1.007 FT 0.943 Blank 16 24 32

[0111] The elution portion with the reaction rate A/A0<90% in Table 9 was significantly competing. The eluent from tubes 10-23 were pooled and dialyzed overnight to prepare for applying to a Phenyl Sepharose Fast Flow column. The protein concentration after dialysis was 5.98 mg/ml in a total volume of 70 ml. Activity of this component: 28 nM/60/0.015 ml?5.95 mg/ml=5.3 U (nM/min/mg).

Phenyl Sepharose Fast Flow:

[0112] 1. Took 20 ml of Phenyl Sepharose Fast Flow packing, packed a column, rinse with deionized water at a flow rate of 10 ml/min for 10 min, and equilibrated with buffer A+220 mM KCl.

[0113] 2. Mixed the sample and the packing in a 4? C. refrigerator using a low speed shaker for 90 minutes.

[0114] 3. Rinsed on the chromatographic system, set UV to zero.

[0115] 4. Eluted with pre-cooled 50% DMSO solution at 6 ml/min. Collected elution peaks and dialyzed the eluent.

Elution Sample Detection:

[0116] Three tubes of the eluent samples were pooled and assayed for MAT activity in the same manner as above with a load of 15 ?l. SAM production was tested as 15 nM. The specific activity of this fraction was 15 nM/60/0.015 ml?2.93 mg/ml=5.7 U (nM/min/mg).

[0117] Due to the limited amount of sample left, no further purification was done. However, MAT activity has been measured after the purification with two chromatography columns. It is suggested that the method of determining MAT activity of the present invention is also applicable to the activity assay of MAT enzyme purified from mouse or rat liver.

Example 7

Effect of Different Procedures on SAM Synthesis

[0118] To clarify the effect of different steps or methods on the amount of SAM production, the MAT enzyme and the substrate were first applied to the PLL-aza-SAM coated strips using the optimal buffer and substrate concentrations described above. The reaction was carried out at 37? C. for 20 min and reacted with HRP-anti-SAM antibody for 40 min. At the same time, MAT was directly reacted with the substrate and HRP-anti-SAM antibody for 1 h. The results showed that the amount of SAM synthesized by the step-by-step or two-step reaction was higher than that produced by the one-step method because the total volume of the system was only 30 ?l during the first 20 min reaction. The concentrations of MAT and the substrates were both 2.33-fold higher than those of the one-step method. Table 10 shows the compared effects of these two different operating procedures on the amount of SAM synthesized. When the MAT concentration was lower at 0.3 mg/ml, the SAM synthesized by the two-step method was 2.81 times higher than that of the one-step method. When the MAT concentration was 0.6 mg/ml, the SAM synthesized by the two-step method was 1.76 times higher than that of the one-step method. When the MAT concentration was increased to 1 mg/ml, the SAM synthesized by the two-step method was 1.42 times higher than that of the one-step method. If MAT concentration continues to increase, it can be foreseen that the stepwise or two-step approach and one-step approach will be similar to each other in the SAM synthesis. The increases in the amount of SAM production (2.81, 1.76, 1.42 times) were not higher than the increases of the substrate and the MAT concentration (3.33 times). The stepwise method was helpful for the improvement of the SAM yield. Especially, in the test when MAT concentration or activity was low, the effects and advantages of the stepwise method would be more pronounced. If the MAT activity of the sample to be tested was high, and it was meant to compare the differences between groups of samples under different conditions, the one-step method is more convenient and easy, and the operating procedures have little influence on results.

TABLE-US-00012 TABLE 10 Effects of different operating procedures on SAM synthesis SAM (nM) MAT Working Concentration (mg/ml) Methods 0.3 0.3 0.6 0.6 1.0 1.0 One-step 189.0947 225.4812 352.0070 392.8603 521.8699 506.2779 Two-step 642.1810 520.4320 628.2861 681.0924 745.7204 713.5748 Avg. fold 2.81 1.76 1.42 increased

[0119] Numerous examples from the present invention demonstrate that the system we have established is very sensitive and can accurately and reliably determine the biological activity of MAT. Although the step-by-step method was used in this example, it was only used to selectively allow the MAT-catalyzed chemical reaction to proceed more efficiently in an independently optimal environment first, and immediately followed by performing quantitative immunological assay. In the second step 40-minute immunoassay, the MAT catalyzed chemical reaction continues to progress. The newly synthesized SAM immediately participates in the competitive immunological reaction. With the optimal system, all synthesized SAM molecules participate in the competition of tracer-labeled anti-SAM antibody. Due to the high specificity of the anti-SAM antibody, the results are very accurate There is no issue of inaccurate measurement caused by SAM loss due to various reasons. Therefore, the present invention proposes that both operating procedures are all very useful and can be used properly based on specific circumstances.

Example 8

Determination of MAT Activity Using Different Coated Antigens and Standards from Above

[0120] In addition to 1 ?g/ml BSA-SAMe that was used for coating the ELISA plate, the same standard SAMe as the coating antigen was used as the standard antigen. HRP-anti-SAM 84-3 (Arthus Biosystems, MAH00202) was used as the anti-SAM antibody. Table 11 shows the result of such an experiment. The linearity of SAMe as a standard product was very good. When MAT was at 0.3 mg/ml, the SAM synthesized was about 3 ?M. The result indicated that the use of SAM analogue aza-SAM plates and standard analogue as well as the SAMe plates and standard can both nicely reflect the changes of MAT activity. Due to differences in binding affinity or other factors, for the same densitometric readings, different amounts of antigens might be required or presented, resulting in differences in the values measured and the linear ranges of the two systems. However, the standard curves are linear and assays are sensitive enough to reflect the changes of MAT activity in both systems described in the present invention. Therefore, both of them may be used.

[0121] Several tests have proved that the results of using the SAMe coated plated and the standard in the present example were generally associated with poor repeatability and unstable linear range compared to those used in Examples 1-7 (coating the SAM analog antigen and using a stable analog as a standard). However, if the source of the raw material SAMe are well controlled and improved, and some related processes and methods are further improved and optimized, the solution in this example is also feasible.

TABLE-US-00013 TABLE 11 Standard curve and results of SAMe for determining MAT activity SAMe (uM) OD450 (SAMe) OD450 (MAT) MAT (mg/ml) 0 1.0328 0.9483 0.3940 0.3 0.3125 1.0548 0.9196 0.4237 0.3 0.625 0.9742 0.9250 0.0448 blank 1.25 0.7431 0.7396 0.0473 2.5 0.4976 0.4788 0.0450 5 0.2461 0.2307 0.0473 10 0.1389 0.1343 0.0453 20 0.1407 0.1447 0.0460

[0122] The above embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the foregoing embodiment, and any other changes, modifications, substitutions, combinations, and other modifications made without departing from the spirit and principle of the present invention which are also possible and included in the present invention. Simplifications should all be equivalent to the replacement methods, and all are included in the protection scope of the claims of the present invention.

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