Affiliation:
1. Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
2. Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark
Abstract
Abstract
Glycogen synthase (GS) is the rate limiting enzyme for glycogen production and together with glycogenin (GN) and glycogen branching enzyme (GBE), can generate glycogen particles containing up to 50,000 glucose units. Dysregulation of glycogen synthesis, for example overproduction or accumulation of misshapen glycogen, is the source of many glycogen storage diseases affecting glucose homeostasis and muscle and neuronal cell function. As such, GS is an attractive candidate enzyme for therapeutic targeting, which until recently, was hampered by difficulties in producing active human GS enzyme preparations. Here, we describe the large-scale production of GS in complex with GN, and assay conditions to measure enzyme activity in the absence and presence of the allosteric activator glucose-6-phosphate (G6P). These protocols, together with assays for quality control assessment of enzyme preparations, provide a useful resource for studying the biochemical, biophysical and structural properties of the GS-GN complex, and facilitate drug discovery pipelines to develop therapeutics for glycogen storage diseases.
Funder
Wellcome Trust
Novo Nordisk Foundation Center for Basic Metabolic Research
Publisher
Research Square Platform LLC
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