CN102221531B - Modified spectrophotometry for detecting activity of HMG-CoA reducase and applications thereof - Google Patents
Modified spectrophotometry for detecting activity of HMG-CoA reducase and applications thereof Download PDFInfo
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Abstract
The invention discloses a modified spectrophotometry for detecting activity of HMG-CoA reducase and applications thereof. The detecting method comprises the following steps: a. preparing liver microsome by centrifuging fresh liver organics; b. carrying out weight drop, homogenate, dilution and centrifugation on the liver microsome, then obtaining a supernatant, and obtaining the reducase source containing dissolvable HMG-CoA; c. detecting the protein content of HMG-CoA reducase source, and diluting the HMG-CoA reducase source to have the known concentration; and d. adding the HMG-CoA reducase source to the optimized reaction system, and detecting the OD340 value and calculating the enzyme activity. The detecting system for HMG-CoA reducase inhibitor is established on the basis of the detecting method and is used for calculating the enzyme activity inhibition rate and half-inhibition concentration (IC50), and can be used for screening hypolipidemic drugs. The modified spectrophotometry has the advantages of system optimization, high safety, low price, batch filtration, credible conclusion and the like, can effectively screen the HMG-CoA reducase activity inhibitor, and provides a simple and quick method for developing the hypolipidemic drugs.
Description
Technical field
The present invention relates to medical technical field, be specifically related to a kind of improvement AAS of the HMG-CoA of mensuration reductase activity, also relate to the application of a kind of HMG-CoA reductase inhibitor detection system in the blood lipid-lowering medicine screening of setting up simultaneously based on this method.
Background technology
Known mevalonic acid can finally generate cholesterol through the reaction of series, and high cholesterol can bring out atherosclerotic, causes diseases such as hypertension, coronary heart disease, thrombus, cerebral hemorrhage, effectively suppresses the generation that the synthetic medicine of cholesterol can reduce these diseases.3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-coenzymeA; HMG-CoA) reductase is the rate-limiting enzyme that mevalonic acid passes through the serial reaction synthetic cholesterol, and levels such as its activity can be synthetic at albumen, degraded and phosphorylation are regulated.All set up though detect the methodology that HMG-CoA reductase mRNA and protein level express in the various regulation mechanisms, estimate protease function finally, effective method must be the detection of enzymatic activity.Known HMG-CoA reductase inhibitor can effectively reduce the cholesterol biosynthesizing, is one type of novel lipid lowerers, also is the atherosclerotic drug of first choice of clinical prevention, and screen and research and develop new HMG-CoA reductase inhibitor is the hot issue in this field always.
The method of classical detection HMG-CoA reductase activity is a radioisotope method, and its principle does
14C mark HMG-CoA, the system reaction is accomplished afterproduct and is done
14The C-mevalonic acid changes it into again
14The C-mevalonolactone separates through the method for chromatography again
14The C-mevalonolactone; Detect its radioactivity; (Kim HK is directly proportional with the HMG-CoA reductase activity; Jeong TS, Lee MK, et a1.Lipid-lowering efficacy of hesperetin metabolites inhigh-cholesterol fed rats.Clinica Chimica Acta.2003; 327:129-37).But because of it uses the radioactive activity material, complex operation needs the pollution of control radiomaterial, and
14The HMG-CoA of C mark and mevalonic acid expensive price, the screening cost is high, brings difficulty therefore for applying of this method.Traditional HMG-CoA reductase activity detects AAS (Ness GC; Spindler CD, Moffler MH.Purification of3-hydroxy-3-methylglutaryl coenzyme A reductase from rat liver.Arch Biochem Biophys.1979; Principle 197:493-9) is: the consumption reflection enzymatic activity of nicotinamide-adenine dinucleotide phosphate in the detection reaction (NADPH), but its specificity is not high, and background (background) significant reaction.In order to reduce background response, need to improve HMG-CoA reductase purity, but bring problems such as step is various, the enzyme yield is lower, so the application of this method is less through follow-up serial purifying.Still do not have both at home and abroad at present through optimizing the mensuration that too many levels in the AAS (like preparation solubility HMG-CoA reductase, pH value of reaction system, zymoprotein amount, concentration of substrate, preparatory temperature bath time and reaction time etc.) carries out the HMG-CoA reductase activity, more do not have the HMG-CoA reductase inhibitor screening system of setting up based on this method.
Summary of the invention
The present invention seeks to be to provide a kind of improvement AAS of the HMG-CoA of detection reductase activity; Centrifugal through homogenate repeatedly, optimize preparatory temperature bath and methods such as reaction time, reaction buffer pH value, protein concentration and concentration of substrate; Obtain the hepatomicrosome of high HMG-CoA reductase activity, improved the specificity and the sensitivity of AAS greatly.
Another object of the present invention is to be to provide a kind of application of HMG-CoA reductase inhibitor detection architecture in the screening blood lipid-lowering medicine based on the improvement AAS.Compare the remarkable cost that reduces screening HMG-CoA reductase inhibitor with the radioisotope method of classics, avoided the use radiomaterial, can be applicable to the screening of serial blood lipid-lowering medicine.
To achieve these goals, the present invention has adopted following technical measures:
A kind of improvement AAS that detects the HMG-CoA reductase activity the steps include:
A, fresh liver tissue (market is purchased) is added homogenate buffer A (100mM sucrose, 50mM KCl, 40mM K with 1: 3 (w/v)
2HPO
4, 30mM EDTA, pH 7.2), homogenate, 12,000 * g is centrifugal twice under the normal temperature, each 15min gets the centrifugal 60min of supernatant 100,000 * g, deposition with homogenate buffer resuspended after, the centrifugal once more 60min of 100,000 * g precipitates and is hepatomicrosome;
B, hepatomicrosome is resuspended with buffer B (buffer A adds the 10mM dithiothreitol (DTT), and pH 7.2) adds 37 ℃ of buffer B that equal-volume contains 50% (v/v) glycerine again; Manually homogenate is after 37 ℃ of temperature are bathed 60min, again with 3 times of buffer B dilutions; Mixing; Last 25 ℃ of centrifugal 60min of 100,000 * g get supernatant, contain the hepatomicrosome albumen of solubility HMG-CoA reductase;
C, detect the protein content that step (B) obtains hepatomicrosome, to 1mg/ml, obtained the known hepatomicrosome albumen of protein concentration with buffer B diluted protein concentration according to protein determination kit;
D, the hepatomicrosome albumen that step (C) is obtained add the reaction system of optimizing (pH 7.0), comprise that NADPH100 μ mol/L, HMG-CoA 50 μ mol/L, protein concentration are 200 μ g/ml and damping fluid C (0.2MKCl, 0.16MK
2HPO
4, 4mMEDTA and 10mM dithiothreitol (DTT)), the reaction cumulative volume is 1ml.Carry out 37 ℃ of temperature bath 20min in advance earlier, add substrate HMG-CoA subsequently to start enzymic catalytic reaction, the reaction time is 60min, uses UV spectrophotometer measuring OD
340The variation of value obtains the HMG-CoA reductase activity according to following formula.
Enzymatic activity [nmol/ (mg.min)]=(measure pipe Δ OD
340-blank Δ OD
340) * 1000/ (6.22 * 0.2 * 60)
According to the HMG-CoA reductase activity that this formula obtains, can be used for calculating the parameters such as inhibiting rate of suppressant, the inhibition that is used for the comparison suppressant is active.
A kind of application of HMG-CoA reductase inhibitor detection architecture in the screening blood lipid-lowering medicine based on the improvement AAS the steps include:
A, by a kind of hepatomicrosome that contains solubility HMG-CoA reductase activity of improvement AAS preparation process gained of the HMG-CoA of detection reductase activity;
B, in above-mentioned HMG-CoA reductase reaction system, add 10 μ l variable concentrations tried thing (HMG-CoA reductase inhibitor), after temperature is bathed 20min in advance, add HMG-CoA and start reaction.
C, measure 60min internal reaction system OD with ultraviolet spectrophotometer
340The variation of value, and according to following formula calculating enzymatic activity inhibiting rate and half-inhibition concentration (IC
50) value.The enzymatic activity inhibiting rate and the IC that obtain according to this formula
50Value can be active big or small to the inhibition of HMG-CoA reductase in order to reflection and comparative drug.
Enzyme inhibition rate (%)=(control tube enzymatic activity-suppressant pipe enzymatic activity)/control tube enzymatic activity * 100
Compare with HMG-CoA reductase inhibitor screening system with existing HMG-CoA reductase activity assay method; Screening system of the present invention has characteristics such as system optimization, safety is inexpensive, batch screens, conclusion is credible; Can be used to accurate and effective screening and have the medicine that suppresses the HMG-CoA reductase activity, easy, detection method fast is provided for developing new fat-reducing medicament.It is specifically:
(1) system optimization: the present invention obtains highly active HMG-CoA reductase source through the repeatedly centrifugal purification to hepatomicrosome, need not to carry out follow-up protein purification process again; Simultaneously, the present invention has carried out optimizing (preparatory temperature bath and reaction time, reaction buffer pH value, protein concentration and concentration of substrate) to the too many levels of reaction, and the specificity of AAS is obviously increased, and the result is more reliable;
(2) safety is inexpensive: compare with the radioisotope method of classics, and non-environmental-pollution, to the "dead" injury of operator, and research cost is extremely low;
(3) screening in batches: the simple, convenient easy row of the screening system of being built can be used for the screening in a short time of medicine in enormous quantities;
(4) conclusion is credible: carry out methodology checking with three kinds of known HMG-CoA reductase inhibitors, also compare with the radioisotope method of classics simultaneously, it is basically identical as a result.Proof can be used for the screening of serial atorvastatin fully based on the HMG-CoA reductase inhibitor detection system of improvement AAS.
Description of drawings
Fig. 1 is the synoptic diagram that influences of the preparatory temperature bath of a kind of difference time HMG-CoA reductase activity
Fig. 2. be the influence synoptic diagram of a kind of differential responses system pH to the HMG-CoA reductase activity
Fig. 3. be the influence synoptic diagram of a kind of differential responses time to the HMG-CoA reductase activity
Fig. 4. be the influence synoptic diagram of a kind of different concentration of substrate to the HMG-CoA reductase activity
Fig. 5. be the influence synoptic diagram of a kind of different microsomal protein amounts to the HMG-CoA reductase activity
Fig. 6. measure the HMG-CoA reductase active synoptic diagram of Pravastatin, Fluvastatin and Rosuvastatin for a kind of the inventive method
A: Pravastatin; B: Fluvastatin; C: Rosuvastatin.
Fig. 7. measure the HMG-CoA reductase active synoptic diagram of Pravastatin, Fluvastatin and Rosuvastatin for a kind of radioisotope method
A: Pravastatin; B: Fluvastatin; C: Rosuvastatin.
Fig. 8. measure the HMG-CoA reductase active synoptic diagram of ginkgo biloba extract GBE50 for a kind of the inventive method.
Embodiment
Through concrete experiment embodiment, further set forth the improvement AAS of measuring the HMG-CoA reductase activity among the present invention and a kind of HMG-CoA reductase inhibitor detection system of setting up based on this method below, and the application in the blood lipid-lowering medicine screening.
Embodiment 1: improvement spectrophotometry HMG-CoA reductase activity
A kind of improvement AAS that detects the HMG-CoA reductase activity the steps include:
A, fresh liver tissue (market is purchased) is added homogenate buffer A (100mM sucrose, 50mM KCl, 40mM K with 1: 3 (w/v)
2HPO
4, 30mM EDTA, pH 7.2), homogenate, 12,000 * g is centrifugal twice under the normal temperature, each 15min gets the centrifugal 60min of supernatant 100,000 * g, deposition with homogenate buffer resuspended after, the centrifugal once more 60min of 100,000 * g precipitates and is hepatomicrosome;
B, hepatomicrosome is resuspended with buffer B (buffer A adds the 10mM dithiothreitol (DTT), and pH 7.2) adds 37 ℃ of buffer B that equal-volume contains 50% (v/v) glycerine again; Manually homogenate is after 37 ℃ of temperature are bathed 60min, again with 3 times of buffer B dilutions; Mixing; Last 25 ℃ of centrifugal 60min of 100,000 * g get supernatant, contain the hepatomicrosome albumen of solubility HMG-CoA reductase;
C, detect the protein content that step (B) obtains hepatomicrosome, to 1mg/ml, obtained the known hepatomicrosome albumen of protein concentration with buffer B diluted protein concentration according to protein determination kit;
D, the HMG-CoA reductase that step C is obtained add reaction system, and [containing NADPH 100 μ mol/L, HMG-CoA 50 μ mol/L, protein concentration is 200 μ g/ml and damping fluid C (0.2M KCl, 0.16MK
2HPO
4, 4mM EDTA and 10mM dithiothreitol (DTT))], the reaction cumulative volume is 1ml, before adding the HMG-CoA reaction, carry out earlier 37 ℃ in advance temperature bathe 20min, add HMG-CoA subsequently and start reaction 60min, use UV spectrophotometer measuring OD
340Change.The result sees Fig. 1-5.
Set up HMG-CoA reductase inhibitor detection architecture based on this detection method, calculate enzymatic activity inhibiting rate and half-inhibition concentration (IC
50), can be used for screening blood lipid-lowering medicine.
Embodiment 2: detection system of the present invention suppresses the conclusive evidence of HMG-CoA reductase activity to Pravastatin, Fluvastatin and Rosuvastatin.
A kind of application of HMG-CoA reductase inhibitor detection architecture in the screening of blood lipid-lowering medicine based on the improvement AAS the steps include:
A, by a kind of hepatomicrosome that contains solubility HMG-CoA reductase activity of improvement AAS preparation process gained of the HMG-CoA of detection reductase activity;
B, set up HMG-CoA reductase inhibitor detection architecture, comprise HMG-CoA reductase (protein content 200 μ g), NADPH (100 μ M), HMG-CoA (50 μ M) and buffer B (0.2M KCl, 0.16M K
2HPO
4, 4mM EDTA and 10mM dithiothreitol (DTT); PH 7.0), add the blood lipid-lowering medicine that has gone on the market (Pravastatin, Fluvastatin and Rosuvastatin) of 10 μ l variable concentrations, the reaction cumulative volume is 1ml; Temperature is bathed 20min in advance, measures 60min internal reaction system OD with ultraviolet spectrophotometer
340Change, and calculate the reductase activity inhibiting rate.
C, result see Fig. 6, and three kinds of medicines all demonstrate good inhibition activity, and are concentration dependent, and related coefficient (R) value is respectively 0.999,0.98,0.87, explains that this result reliability is high.Through using the HMG-CoA reductase active that method of the present invention detects three kinds of blood lipid-lowering medicines that gone on the market, set up the inhibitor sifting method, obtained best screening reaction conditions.
Embodiment 3: the radioisotope method of detection system of the present invention and HMG-CoA reductase activity relatively.
The radioisotope method of detection system of the present invention and HMG-CoA reductase activity relatively the steps include:
A, contain the hepatomicrosome of solubility HMG-CoA reductase by the said methods preparation of embodiment 2;
B, set up HMG-CoA reductase inhibitor detection architecture, detect the half-inhibition concentration (IC of the blood lipid-lowering medicine (Pravastatin, Fluvastatin and Rosuvastatin) that has gone on the market by embodiment 2
50).The result sees table 1, the IC of Pravastatin, Fluvastatin and Rosuvastatin
50Be respectively 11.07,6.19 and 3.19 μ g/L.
C, employing radioisotope method (Kim HK, Jeong TS, Lee MK, et al.Lipid-lowering efficacy ofhesperetin metabolites in high-cholesterol fed rats.Clinica Chimica Acta 2003; 327:129-37), detect the IC of Pravastatin, Fluvastatin and Rosuvastatin
50, the HMG-CoA reductase detection system that the present invention set up is verified.The result sees Fig. 7 and table 1, the IC of Pravastatin, Fluvastatin and Rosuvastatin
50Be respectively 14.57,19.96 and 5.74 μ g/L, the HMG-CoA reductase detection system result who is set up with the present invention is more consistent, thereby proves HMG-CoA reductase detection system reliable results of the present invention, has repeatability.
Table 1. improvement AAS and radioisotope method detect Pravastatin, Fluvastatin and Rosuvastatin
Half-inhibition concentration (IC to the HMG-CoA reductase
50).
Embodiment 4: detection system of the present invention is screened multiple blood lipid-lowering medicines such as ginkgo biloba extract GBE50.
A kind of application of HMG-CoA reductase inhibitor detection architecture in the screening blood lipid-lowering medicine based on the improvement AAS the steps include:
A, by a kind of hepatomicrosome that contains solubility HMG-CoA reductase activity of preparation process gained of improvement AAS of the HMG-CoA of detection reductase activity;
B, set up HMG-CoA reductase detection architecture, comprise HMG-CoA reductase enzyme protein amount 200 μ g, NADPH (100 μ M), HMG-CoA (50 μ M) and buffer C (0.2M KCl, the 0.16M K of embodiment 1 preparation
2HPO
4, 4mM EDTA and 10mM dithiothreitol (DTT), pH 7.0), and add 10 μ l variable concentrations GBE50, the reaction cumulative volume is 1ml, temperature is bathed 20min in advance, measures 60min internal reaction system OD with ultraviolet spectrophotometer
340Change, calculate reductase activity inhibiting rate and IC
50Be worth, receive the action intensity of reagent thing in order to reflection.
C, result see Fig. 8.Confirm that Chinese medical extract GBE50 can effectively suppress the HMG-CoA reductase activity, and inhibiting effect is the dose dependent enhancing.This and this chamber records the inhibiting basically identical as a result of HMG-CoA reductase activity (Xie ZQ, the et al.Molecular mechanisms underlyingthe cholesterol-lowering effect of Ginkgo biloba extract in hepatocytes:a comparativestudy with lovastatin.Acta Pharmacol Sin 2009 of GBE50 early stage with radioisotope method; 30:1262-75).
Claims (2)
1. an improvement AAS that detects the HMG-CoA reductase activity the steps include:
A, with the fresh liver tissue with 1: 3w/v adds homogenate buffer A:100mM sucrose, 50mM KCl, 40mMK
2HPO
4, 30mM EDTA, pH 7.2, homogenate, 12,000 * g is centrifugal twice under the normal temperature, each 15min gets the centrifugal 60min of supernatant 100,000 * g, deposition with homogenate buffer resuspended after, the centrifugal once more 60min of 100,000 * g precipitates and is hepatomicrosome;
B, hepatomicrosome is used buffer B: buffer A adds the 10mM dithiothreitol (DTT), and pH 7.2 is resuspended, adds 37 ℃ of buffer B that equal-volume contains 50%v/v glycerine again; Manually homogenate is after 37 ℃ of temperature are bathed 60min, again with 3 times of buffer B dilutions; Mixing; Last 25 ℃ of centrifugal 60min of 100,000 * g get supernatant, contain the hepatomicrosome albumen of solubility HMG-CoA reductase;
C, detect the protein content that step B obtains hepatomicrosome, to 1mg/ml, obtained the known hepatomicrosome albumen of protein concentration with buffer B diluted protein concentration according to protein determination kit;
D, the hepatomicrosome albumen that step C is obtained add the reaction system of optimizing and obtain improveing the AAS detection architecture; This improvement AAS detection architecture pH 7.0; Comprise that NADPH 100 μ mol/L, HMG-CoA 50 μ mol/L, protein concentration are 200 μ g/ml and damping fluid C:0.2M KCl, 0.16M K
2HPO
4, 4mMEDTA and 10mM dithiothreitol (DTT); The reaction cumulative volume is 1ml, before adding the HMG-CoA reaction, carries out 37 ℃ of temperature bath 20min in advance earlier; Add substrate HMG-CoA subsequently to start enzymic catalytic reaction; Reaction time is 60min, with the variation of UV spectrophotometer measuring OD340 value, obtains the HMG-CoA reductase activity according to following formula;
Enzymatic activity [nmol/ (mg.min)]=(measure pipe Δ OD
340-blank Δ OD
340) * 1000/ (6.22 * 0.2 * 60).
2. the application of improvement AAS detection architecture in the screening of blood lipid-lowering medicine that improvement AAS according to claim 1 is set up the steps include:
A, make the hepatomicrosome albumen that contains solubility HMG-CoA reductase by the improvement AAS preparation process of the described detection of claim 1 HMG-CoA reductase activity;
B, in the improvement AAS detection architecture described in the claim 1 step D, add the HMG-CoA reductase inhibitor of 10 μ l variable concentrations, before adding the HMG-CoA reaction, carry out earlier 37 ℃ in advance temperature bathe 20min, add HMG-CoA subsequently and start reaction;
C, measure 60min internal reaction system OD with ultraviolet spectrophotometer
340The variation of value, and according to following formula calculating enzyme inhibition rate, can be active big or small to the inhibition of HMG-CoA reductase according to enzyme inhibition rate and half-inhibition concentration value that this formula obtains in order to reflection and comparative drug;
Enzyme inhibition rate (%)=(control tube enzymatic activity-suppressant pipe enzymatic activity)/control tube enzymatic activity * 100.
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| CN103243147A (en) * | 2012-02-09 | 2013-08-14 | 香港浸会大学 | Novel high-throughput yeast cell-based screening platform for identifying HMG-CoA reductase inhibitors and uses of such inhibitors |
| CN103411928B (en) * | 2013-07-18 | 2015-10-28 | 湖南大学 | The method of detection fibers disaccharidase inhibitor |
| CN104651437A (en) * | 2015-03-07 | 2015-05-27 | 刘红卫 | Blood fat reducing active peptide separated from codium fragile and application thereof |
| CN107271384A (en) * | 2017-06-08 | 2017-10-20 | 广州金域医学检验中心有限公司 | Method for detecting activity of erythrocyte dihydropteridine reductase, detection kit and application thereof |
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| CN101270385A (en) * | 2007-03-21 | 2008-09-24 | 中国科学院沈阳应用生态研究所 | Analysis method for testing soil denitrification enzyme liveness |
| CN101308119A (en) * | 2008-04-16 | 2008-11-19 | 上海市徐汇区中心医院 | Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity |
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| JPH10185687A (en) * | 1996-12-24 | 1998-07-14 | Shimadzu Corp | Enzyme activity value measuring device using spectrophotometer |
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| CN101270385A (en) * | 2007-03-21 | 2008-09-24 | 中国科学院沈阳应用生态研究所 | Analysis method for testing soil denitrification enzyme liveness |
| CN101308119A (en) * | 2008-04-16 | 2008-11-19 | 上海市徐汇区中心医院 | Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity |
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