Role of enzyme interactions in the regulation of glycolysis and gluconeogenesis. Purification and properties of the phospho- and dephospho-forms of glycogen phosphorylase from swine kidney.

Dephosphophosphorylase was isolated from swine kidney by a procedure involving precipitation with acetone and ethanol in the presence of glycogen, ammonium sulfate fractionation and chromatography on DEAE-cellulose. The enzyme was purified about 15000-fold to a final specific activity of 30 μmol glucose-1-phosphate formed from glycogen and inorganic phosphate per min per mg protein at 26°C. Yields of 20% of the activity present in crude extracts were obtained by this method. The purified enzyme could be completely converted to a phosphorylated form by incubation with ATP, magnesium ion and a partially purified soluble protein kinase which was isolated from the same crude kidney extracts. The final specific activity of phosphophosphorylase isolated by the same procedure was about 70 μmol glucose-1-phosphate formed from glycogen and inorganic phosphate per min per mg protein at 26°C. Ultracentrifugation and polyacrylamide-gel electrophoresis studies with the purified enzymes indicated that the preparations were homogeneous. Molecular weight determination by Sephadex G-200 and Sepharose 6-B column chromatography revealed V0/Ve ratios which corresponded to a molecular weight of 190000 ± 5000 for both of the enzyme preparations. Sucrose-density centrifugation further confirmed that both forms of kidney phosphorylase had similar molecular weights of about 190000 ± 10000 and s20,w values of from 8.1 to 8.3 S. The s20,w values obtained in ultracentrifugation studies, 8.3 to 8.4 S, agreed reasonably well with those obtained by sucrose density centrifugation. The molecular weights of the two forms of the enzyme calculated from this data and a diffusion coefficient of 0.4 μm2/s which was determined by exclusion chromatography and a partial specific volume of 0.735 cm3/g was 195000 ± 5000 in each case. When the enzymes were subjected to polyacrylamide-gel electrophoresis in the presence of sodium dodecyl-sulfate a single sharp protein band was observed. These studies showed that both forms of the enzyme contained two identical subunits with molecular weights of 95000. Some of the kinetic and allosteric properties of the phospho and dephospho-forms of purified kidney phosphorylase were examined and compared. The Km of the phosphorylated form of the enzyme for Pi was 2.8 mM compared to 100 mM for the dephospho-form in the presence of 0.2 mg glycogen and 2 mM AMP. The corresponding values for glycogen at about 4 mM Pi were 1.4 mM and 20 mM for the phospho and dephospho-forms, respectively. The apparent Km values for Pi or glycogen varied as a function of the other substrate for the dephospho-enzyme. This effect was not observed with the phospho-enzyme. The dephospho-form of kidney phosphorylase required AMP for activity and protamine also stimulated the activity of this form of the enzyme. Glucose (0.5 mM) inhibited the activity of kidney dephosphophosphorylase. A protein kinase isolated from crude kidney extracts converted the dephospho-form of the enzyme to a phosphorylated form in the presence of ATP and magnesium. About two equivalents of phosphate were transferred from ATP to the phosphorylated form of the enzyme during this conversion.