Role of Enzyme-Enzyme Interactions in the Regulation of Glycolysis and Gluconeogenesis PROPERTIES OF GLYCOGEN SYNTHETASE ISOLATED FROM SWINE KIDNEY

Abstract 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 10,000-fold to a final specific activity of 9.1 µmoles of glucose transferred from UDP-d-glucose 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 mm 2-mercaptoethanol. The final preparation was essentially homogeneous as determined by polyacrylamide gel electrophoresis, elution profiles from DEAE-cellulose, Bio-Gel A1.5 and Sepharose 6B columns, and sucrose density gradient centrifugation. 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 kinase, phosphorylase, and protein phosphatases when measured with phosphorylase a, phosphoglucomutase, casein, phosvitin, and glycogen synthetase labeled with 32P 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% sodium dodecyl sulfate which showed that the enzyme contained 4 subunits with molecular weights of 92,000 ± 3,000. Some of the kinetic properties of the active and inactive forms of kidney glucogen synthetase were examined. Glucose-6-P stimulated the activity of the inactive form of the enzyme. There was no activity in the absence of glucose-6-P and the Km for UDP-glucose was 1.1 x 10-4 m at 1 mm glucose-6-P and 6.7 x 10-5 m at 11 mm glucose-6-P. The Km of the active form of the enzyme for UDP-glucose was 8.7 x 10-4 m, and the activity was increased only slightly in the presence of glucose-6-P. The Km of the active form of the enzyme for UDP-glucose was about 15 times greater than that of the inactive form. The purified enzyme preparation was completely free of glycogen, and no activity was observed in the absence of this primer. Increasing concentrations of glycogen affected the activity of both forms of the enzyme in a similar manner.