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Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism
Published on: August 19, 2021
An enzyme-coupled continuous spectrophotometric assay for glycogen synthases
Nahuel Z Wayllace1, Hugo A Valdez, Andrea Merás
1Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina.
Molecular Biology Reports
|May 18, 2011
Summary
A new continuous spectrophotometric assay quantifies bacterial glycogen synthase (GS) activity using ADP-glucose or UDP-glucose. This method enables efficient determination of kinetic parameters for GS enzymes.
Area of Science:
- Biochemistry
- Enzymology
- Microbial Metabolism
Background:
- Bacterial glycogen synthesis involves enzymes like glycogen synthase (GS), which elongates α-1,4-glucan.
- Agrobacterium tumefaciens GS utilizes ADPGlc preferentially but can use UDPGlc less efficiently.
Purpose of the Study:
- To develop a continuous spectrophotometric assay for determining bacterial glycogen synthase (GS) activity.
- To enable the use of both ADPGlc and UDPGlc as glycosyl donors in the assay.
Main Methods:
- Coupling ADP production to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH) for ADPGlc substrate.
- Converting UDP to ADP via adenylate kinase, then coupling to PK and LDH for UDPGlc substrate.
- Improved expression of A. tumefaciens GS in Escherichia coli BL21(DE3)-RIL cells.
Main Results:
- The developed assay allows continuous monitoring of glycosyltransferase activity.
- Kinetic parameters of A. tumefaciens GS were determined using the new assay.
- Results were compared with those from the classical radiochemical method.
Conclusions:
- The continuous spectrophotometric assay provides an efficient method for assessing bacterial GS activity.
- The assay is versatile, accommodating both ADPGlc and UDPGlc as substrates.
- Optimized GS expression facilitates kinetic studies.
