Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay |
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Authors: | Snia Rocha Mariana Lucas Alberto N Araújo M Luísa Corvo Eduarda Fernandes Marisa Freitas |
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Institution: | 1.LAQV-REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; (S.R.); (M.L.); (A.N.A.);2.Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa, 1649-003 Lisboa, Portugal; |
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Abstract: | 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 °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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Keywords: | glycogen phosphorylase optimization enzyme activity type 2 diabetes |
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