Purification and characterization of a cAMP- and Ca2+-calmodulin-independent glycogen synthase kinase from porcine renal cortex.
We recently reported the partial purification of a cAMP-independent and Ca2+-calmodulin-independent glycogen synthase kinase from porcine renal cortex (Schlender, K. K., Beebe, S. J., and Reimann, E. M. (1981) Cold Spring Harbor Conf. Cell Proliferation, 389-400). Subsequent purification indicated that the enzyme preparation consisted of at least three forms of glycogen synthase kinase which could be resolved by ATP gradient elution from aminoethylphosphate-agarose (AEP-agarose). The predominant form of glycogen synthase kinase, which eluted from AEP-agarose between 2 and 6 mM ATP, was purified approximately 800-fold and is designated GSK-A1. It had a molecular weight of 45,000-50,000 as determined by gel filtration and sucrose density gradient centrifugation. It catalyzed the transfer of 1 mol of 32P/mol of synthase subunit into a low molecular weight (10,000) CNBr peptide which was tentatively identified as Ser-7 (site 2) by high performance liquid chromatography. This phosphorylation decreased the activity ratio (activity in the absence of glucose-6-P divided by activity in the presence of 7.2 mM glucose-6-P) from 0.95 to about 0.55. GSK-A1 appeared to be specific for and had low s0.5 values for both substrates, ATP (13 microM) and glycogen synthase (0.3-0.4 microM). The enzyme could not use GTP as the phosphate donor. GSK-A1 was not affected by the protein kinase inhibitor, cAMP, cGMP, Ca2+-calmodulin, EGTA, or trifluoperazine and had a broad pH optimum (pH 7.0-8.5). A second form, GSK-A2, was eluted from AEP-agarose between 7 and 9 mM ATP. GSK-A2 could transfer a 2nd mol of 32P/mol of synthase subunit and decreased the activity ratio to 0.30. The interrelation among these multiple forms is not clear, but the data suggest that multiple kinases are required to form the highly inactivated glycogen synthase in renal tissues.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000378:metabolism; D038341:Glycogen Synthase Kinases; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013552:Swine
D002147:Calmodulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D000242:Cyclic AMP; D006006:Glycogen Synthase; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium