Glycogen synthase I was purified from human polymorphonuclear leukocytes by a procedure involving affinity chromatography of the glycogen-enzyme complex, digestion of endogenous glycogen by amylase, starch chromatography and gel filtration. The purified enzyme had a specific activity of 7–11 U/mg protein, or 4–5 U when expressed per mg of residual glycogen. Further purification to 21 U/mg protein could be achieved. The enzyme was inactive in the absence of added glycogen. A subunit molecular weight of 85000 was determined by polyacrylamide electrophoresis in sodium dodecylsulfate. The molecular weight of the native enzyme was estimated to be 390000 (13.2 S) by sucrose gradient centrifugation and 410000 by gel filtration indicating that the native enzyme is a tetramer. The gel filtration behavior was not affected by enzyme concentration, temperature, or the presence of ligands. The energy of activation was estimated to 13500 cal/mol (56.5 kJ/mol), corresponding to a Q10 of 2.2. In the presence of glucose 6-phosphate or Na2SO4, the enzyme showed a broad pH optimum between pH 6.8–9.2. In the absence of these ligands and in particularly in the presence of Mg2+, the enzyme is sensitive to small changes of pH in the interval pH 7.4–8.4.
During purification, synthase I requires protection by 0.6 mM dithiothreitol, while high concentrations of mercaptoethanol or dithiothreitol inactivates the enzyme, particularly during freezing. During 24-h incubations, synthase I undergoes a spontaneous, temperature-dependent inactivation which is not due to proteolysis, but presumably is caused by irreversible conformational changes. These can be prevented by high concentrations of glucose 6-phosphate, Na2SO4, inorganic phosphate, UDP and glycogen. Mg2+ and traces of ethanol inactivates the enzyme. The lyophilized enzyme is stable for years.
Glycogen synthase (EC 188.8.131.52)