期刊
MOLECULES
卷 26, 期 15, 页码 -出版社
MDPI
DOI: 10.3390/molecules26154635
关键词
glycogen phosphorylase; optimization; enzyme activity; type 2 diabetes
资金
- FCT/MCTES through national funds [UID/QUI/50006/2020]
- [PTDC/NAN-MAT/29248/2017-POCI-01-0145-FEDER-029248]
The study presents a colorimetric method for measuring the activity of glycogen phosphorylase, providing optimal parameters for enzyme concentration, substrate concentration, and temperature. The method was validated using known inhibitors and can be applied for high-throughput screening of potential GP inhibitors in the future.
Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 degrees C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.
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