Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Sónia Rocha,M Luísa Corvo,Marisa Freitas,Alberto N Araújo,Mariana Lucas,Eduarda Fernandes

doi:10.3390/molecules26154635

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.

Highlights

Glycogen phosphorylase (GP) (E.C.2.4.1.1) is a key enzyme in the glycogenolysis pathway
In order to optimize the experimental variables for GP inhibitory studies, several parameters were evaluated, including GPa amount (Figure 5), glucose 1-phosphate (Figure 6), and glycogen concentrations (Figure 7), and the dependence of the enzyme activity on temperature (Figure 8)
The experimental parameters found in the literature to measure GP activity are highly variable, and standardization of these parameters becomes essential to enable a correct reproduction in any laboratory, allowing a comparison of the reported results

Summary

Introduction

Glycogen phosphorylase (GP) (E.C.2.4.1.1) is a key enzyme in the glycogenolysis pathway. A high degree of homology (≈80%) in the amino acid sequences was found among the three isoforms [7]. These isoforms are regulated allosterically by diverse molecule effectors and by reversible phosphorylation of serine-14 through the involvement of phosphorylase kinase (PhK), and dephosphorylation by protein phosphatase 1 (PP1). It has been described in two interconvertible forms, a phosphorylated form, GPa, and a dephosphorylated form, GPb, but only the phosphorylated form is catalytically active [5,8].

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