Phosphorylation of heart glycogen synthase by cAMP-dependent protein kinase. Regulatory effects of ATP.
Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation; D006006:Glycogen Synthase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology
D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D006006:Glycogen Synthase; D011494:Protein Kinases