Modified spectrophotometry for detecting activity of HMG-CoA reducase and applications thereof

A technology of spectrophotometry and reductase, which is applied in the field of medicine, can solve problems such as numerous steps, low specificity, and cumbersome operations, and achieve the effects of no radiation damage, increased specificity, and low research costs

Inactive Publication Date: 2011-10-19
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because of the use of radioactive substances, the operation is cumbersome, and the pollution of radioactive substances needs to be controlled, and 14 C-labeled HMG-CoA and mevalonate are expensive, and the screening cost is high, which brings difficulties to the popularization and application of this method
Traditional HMG-CoA reductase activity detection spectrophotometry (Ness GC, Spindler CD, Moffler MH. Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat liver. Arch Biochem Biophys.1979; 197: 493-9) The principle is: the consumption of nicotinamide adenine dinucleotide phosphate (NADPH) in the detection reaction reflects the enzyme activity, but its specificity is not high, and the background (background) reaction is obvious
In order to reduce the background reaction, it is necessary to improve the purity of HMG-CoA reductase through a series of subsequent purifications, but it brings problems such as many steps and low enzyme yield, so the application of this method is seldom
At present, there is no method of HMG-CoA reductase at home and abroad by optimizing multiple steps in the spectrophotometric method (such as preparation of soluble HMG-CoA reductase, pH value of the reaction system, enzyme protein amount, substrate concentration, pre-incubation time and reaction time, etc.) Determination of activity, not to mention the HMG-CoA reductase inhibitor screening system established based on this method

Method used

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  • Modified spectrophotometry for detecting activity of HMG-CoA reducase and applications thereof
  • Modified spectrophotometry for detecting activity of HMG-CoA reducase and applications thereof
  • Modified spectrophotometry for detecting activity of HMG-CoA reducase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Improved spectrophotometric assay for HMG-CoA reductase activity

[0036] An improved spectrophotometric method for detecting HMG-CoA reductase activity, the steps of which are:

[0037] A. Add fresh liver tissue (purchased in the market) into homogenization buffer A (100mM sucrose, 50mM KCl, 40mM K 2 HPO 4 , 30mM EDTA, pH 7.2), homogenate, centrifuge twice at 12,000×g at room temperature, 15min each time, take the supernatant and centrifuge at 100,000×g for 60min, resuspend the pellet with homogenization buffer, and centrifuge again at 100,000×g for 60min, Precipitation is liver microsomes;

[0038] B. Resuspend the liver microsomes with buffer B (buffer A plus 10mM dithiothreitol, pH 7.2), then add an equal volume of 37°C buffer B containing 50% (v / v) glycerol, and homogenize manually After incubating at 37°C for 60 minutes, dilute 3 times with buffer B, mix well, and finally centrifuge at 100,000×g at 25°C for 60 minutes to get the supernatant, which co...

Embodiment 2

[0042] Example 2: Confirmation that the detection system of the present invention inhibits HMG-CoA reductase activity by pravastatin, fluvastatin and rosuvastatin.

[0043] An application of an HMG-CoA reductase inhibitor detection system based on improved spectrophotometry in the screening of blood lipid-lowering drugs, the steps of which are:

[0044] A, liver microsomes containing soluble HMG-CoA reductase activity obtained by an improved spectrophotometric method for detecting HMG-CoA reductase activity;

[0045] B. Establish a detection system for HMG-CoA reductase inhibitors, including HMG-CoA reductase (200 μg protein), NADPH (100 μM), HMG-CoA (50 μM) and buffer B (0.2M KCl, 0.16M K 2 HPO 4 , 4mM EDTA and 10mM dithiothreitol, pH 7.0), add 10μl of different concentrations of marketed lipid-lowering drugs (pravastatin, fluvastatin and rosuvastatin), the total reaction volume is 1ml, pre-warming bath for 20min , use a UV spectrophotometer to measure the OD of the reactio...

Embodiment 3

[0047] Example 3: Comparison of the detection system of the present invention with the radioisotope method of HMG-CoA reductase activity.

[0048] Detection system of the present invention is compared with the radioisotope method of HMG-CoA reductase activity, and its steps are:

[0049] A. Prepare liver microsomes containing soluble HMG-CoA reductase according to the method described in Example 2;

[0050] B, establish HMG-CoA reductase inhibitor detection system according to embodiment 2, detect the half maximal inhibitory concentration (IC) of the hypolipidemic drug (pravastatin, fluvastatin and rosuvastatin) that has been listed 50 ). The results are shown in Table 1, IC of pravastatin, fluvastatin and rosuvastatin 50 They were 11.07, 6.19 and 3.19 μg / L, respectively.

[0051] C. Using radioisotope method (Kim HK, Jeong TS, Lee MK, et al. Lipid-lowering efficacy of hesperetin metabolites in high-cholesterol fed rats. Clinica Chimica Acta 2003; 327: 129-37) to detect pra...

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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 [0001] The present invention relates to the field of medical technology, in particular to an improved spectrophotometric method for measuring the activity of HMG-CoA reductase, and also relates to the application of an HMG-CoA reductase inhibitor detection system established based on the method in the screening of blood lipid-lowering drugs application. Background technique [0002] It is known that mevalonate can eventually generate cholesterol through a series of reactions. High cholesterol can induce atherosclerosis, leading to hypertension, coronary heart disease, thrombosis, cerebral hemorrhage and other diseases. Drugs that effectively inhibit cholesterol synthesis can reduce the occurrence of these diseases. 3-hydroxy-3-methylglutaryl-coenzymeA (3-hydroxy-3-methylglutaryl-coenzymeA, HMG-CoA) reductase is the rate-limiting enzyme for mevalonate to synthesize cholesterol through a series of reactions, and its activity can be detected in protein Synthesi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33C12Q1/26
Inventor 汪晖郭喻平洁梁赅王婷婷
Owner WUHAN UNIV
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