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A kind of myosin ATP enzyme activity micro-assay method and its application

A technique for myosin and micro-measurement, which is used in organic active ingredients, pharmaceutical formulations, medical preparations containing active ingredients, etc. accurate effect

Inactive Publication Date: 2011-12-21
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The invention can stabilize the color of monomeric phosphorus produced by myosin hydrolyzing ATP within 10 minutes, and overcomes the problem of inaccurate quantification caused by slow color development in the classical method

Method used

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  • A kind of myosin ATP enzyme activity micro-assay method and its application
  • A kind of myosin ATP enzyme activity micro-assay method and its application
  • A kind of myosin ATP enzyme activity micro-assay method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Determination of the inhibition rate of blebbistatin on myosin ATPase

[0049] Take 2 μl of myosin, 10 μl of calcium chloride solution, 2 μl of blebbistatin dilution (1, 3, 5, 7 μM respectively), add Tris-maleic acid buffer (25 mM, pH 7.0) to a final volume of 100 μl, mix Incubate in a water bath at 25°C for 10 minutes. Add 5 μl of ATP, mix well and incubate in a water bath at 25°C for 10 min, then add 100 μl of 15% TCA to stop the reaction. After centrifugation, 60 μl of the supernatant was taken and placed in a 96-well plate, and after adding 20 μl of ammonium molybdate solution and 8 μl of stannous chloride solution for color development, the absorbance was measured at 700 nm. Table 2 results show that the enzyme concentration is 0.1mg.ml -1 , the substrate concentration was 0.5mM, and the reaction was carried out in a water bath at 25°C for 10 minutes. The IC of blebbistatin on the inhibition of myosin ATPase activity was measured. 50 3.07μmol.L -1 , which is si...

Embodiment 2

[0054] The determination method is the same as that in Example 1, and the concentration of ruscogenin is 10, 15, 20, 25 μM.

[0055] Table 3 Inhibitory effect of ruscogenin on myosin II ATPase

[0056]

[0057] Notice: ＊ P＊＊ P<0.01 compared with solvent control group

[0058] This method was used to screen myosin ATPase activity inhibitors, and it was found for the first time that ruscogenin had strong inhibitory activity, IC 50 22.75μmol.L -1 . In view of the literature reports that inhibitors of myosin ATPase activity can inhibit tumor cell migration, regulate renal function, inhibit retinal degeneration, cell death, etc. Advances in Functional Research. Advances in Modern Biomedicine, 2009, 9(20): 3964-3979; Manzanares MV et al. Non-muscle myosin II takes center stage in cell adhesion and migration. Nature Reviews Molecular Cell Biology, 2009, 10(11 ): 778-790; Wrighton KH. Autophagy: Myosin II moves in on autophagosomes. Nature Reviews Molecular Cell Biology, 201...

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Abstract

The invention relates to a method for the microdetermination of ATPase activity of myoglobulin, and the method is characterized in that based on the reaction method of a classic molybdenum blue method, the molar ratio of ATP (adenosine-triphosphate) to myoglobulin is controlled at 2000:1 and the molar ratio of ammonium molybdate to stannous chloride is controlled at 5.5:1. The determination method provided by the invention is simple, convenient, fast, high in response value and wide in linear range, and can be applied to the screening of inhibitors. The invention discloses an application of Ruscogenin in inhibiting the ATPase activity of the myoglobulin.

Description

technical field [0001] The invention relates to a micro-measurement method of myosin ATPase activity, which is based on the reaction principle of the classic molybdenum blue method, through the investigation of influencing factors such as substrate concentration, pH value of the reaction system, calcium ion concentration, and reaction time, to determine the optimal method. Reaction conditions, the method is simple, rapid, high response value, wide linear range, and can be applied to the screening of inhibitors, and the use of ruscogenin in inhibiting the activity of myosin ATPase is found. Background technique [0002] Myosin was first discovered in the muscle tissue and cytoplasm of animal cells. According to different sources, it can be divided into traditional myosin and non-traditional myosin. Myosin is involved in a variety of cellular activities including muscle contraction, cytokinesis, synapse growth, targeted vesicle transport and signal transduction, and is a very ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78A61K31/58
Inventor 寇俊萍张冠军余伯阳
Owner CHINA PHARM UNIV
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