Purification and characterization of rabbit liver calmodulin-dependent glycogen synthase kinase.
A rabbit liver cAMP-independent glycogen synthase kinase has been purified 4500-fold to a specific activity of 2.23 mumol of 32P incorporated per min per mg of protein using ion exchange chromatography on DEAE-Sephacel and phosphocellulose, gel filtration chromatography on Sepharose 6B, and affinity chromatography on calmodulin-Sepharose. This synthase kinase, which was completely dependent on the presence of calmodulin (apparent K0.5 = 0.1 microM) and calcium for activity, also catalyzed the phosphorylation of purified smooth muscle myosin light chain but not of smooth muscle myosin. Using 0.5 mM ATP, a maximal rate of phosphorylation of glycogen synthase was achieved in the presence of 10 mM magnesium acetate with a pH optimum of 7.8. Gel filtration experiments indicated a Stokes radius of about 70 A and sucrose density gradient centrifugation data gave a sedimentation coefficient of 10.6 S. A molecular weight of approximately 300,000 was calculated. A definitive subunit structure was not determined, but major bands observed after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponded to a doublet at 50,000 to 53,000. The calmodulin-dependent glycogen synthase kinase incorporated about 1 mol of 32P per mol of synthase subunit into sites 2 and 1b associated with a decrease in the synthase activity ratio from 0.8 to about 0.4. The calmodulin-dependent glycogen synthase kinase may mediate the effects of alpha-adrenergic agonists, vasopressin, and/or angiotensin II on glycogen synthase in liver.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D038341:Glycogen Synthase Kinases; D008099:Liver / Q000201:enzymology*; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D011817:Rabbits; D013379:Substrate Specificity
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009218:Myosins