Role of enzyme-enzyme interactions in the regulation of glycolysis and gluconeogenesis. Properties of glycogen synthetase isolated from wine kidney.

Glycogen synthetase was extensively purified from swine kidney by a procedure involving adsorption to calcium phosphate gel, ammonium sulfate fractionation, precipitation with ethanol, and chromatography on DEAE-cellulose. The enzyme was purified more than lO,OOO-fold to a final specific activity of 9.1 pmoles of glucose transferred from UDP-Dglucose to glycogen per min per mg of protein at 37’. Yields of 15 to 30% of the activity present in the crude extracts were consistently obtained by this isolation procedure. The purified enzyme was converted to an inactive form by washed kidney particulate preparations. When a step involving incubation in the presence of these particulate fractions, ATP, and magnesium ion was included in the purification procedure, only the form of glycogen synthetase dependent on the presence of glucose 6-phosphate for activity was isolated. The stability of the enzyme at all stages of purification was greatly increased in the presence of 0.3 M sucrose and 20 mu 2-mercaptoethanol. The final preparation was essentially homogeneous as determined by polyacrylamide gel electrophoresis, elution profiles from DEAE-cellulose, Bio-Gel Al.5 and Sepharose 6B columns, and sucrose density gradient centrlfugation. The purified preparation was free of protein kinases which convert glycogen synthetase to an inactive form. The enzyme was also completely free of phosphofructokinase, phosphorylase lrinase, phosphorylase, and protein phosphatases when measured with phosphorylase a, phosphoglucomutase, casein, phosvitin, and glycogen synthetase labeled with 3ZP as substrates. The molecular weight of kidney glycogen synthetase calculated from data obtained by sucrose density centrifugation and chromatography on Sepharose 6B was 370,000. Analysis of the amino acid composition indicated a high ratio of acidic to basic residues, which may account in part for the tight binding of the enzyme to DEAE-cellulose columns. Sedimentation equilibrium analysis of the denatured enzyme in 4 M guanidine hydrochloride indicated that four polypeptide chains were present in the native enzyme. These results were confirmed by polyacrylamide gel electrophoresis in 0.1%