Spectrophotometric Assay to Determine Glycogen Synthase Activity

Glycogen synthase ― a crucial enzyme in glycogen synthesis ― cleaves uridine diphosphate  glucose, or UDP-glucose ― a nucleotide sugar ― into UDP and glycosyl residue and adds the released glucose monomer to the growing glycogen chain.

To estimate glycogen synthase activity, begin by taking a reaction mixture containing glycogen, UDP-glucose, ATP, phosphoenolpyruvate, and nicotinamide adenine dinucleotide, or NADH, in a tube. Add nucleoside diphosphate, or NDP, kinase enzyme, and, a pyruvate kinase and lactate dehydrogenase enzyme cocktail.  Mix the contents, and incubate.

Add glycogen synthase-containing sample to the tube to initiate the enzymatic reaction.

Glycogen synthase acts on UDP-glucose, generating UDP and a glycosyl residue. The enzyme takes up the free glucose monomer and incorporates it into the reaction mixture's glycogen chain. In the presence of ATP, the reaction mixture's NDP kinase phosphorylates the unused UDP, converting it into UTP and generating ADP.

The enzyme pyruvate kinase acts on ADP and adds a phosphate donated by the reaction mixture's phosphoenolpyruvate to produce ATP and pyruvate. Next, lactate dehydrogenase converts the pyruvate to lactate and oxidizes NADH during the reaction.

Measure the decrease in NADH absorbance at 340 nm to quantify the amount of NADH consumed, which indicates successful enzyme activity.