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Glycogen synthase kinase 3 was purified 80-100000-fold from extracts of rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate, chromatography on phosphocellulose and Affigel Blue, and affinity chromatography on (glycogen synthase)-Agarose. 0.08 mg of protein was isolated from 5000 g muscle corresponding to a yield of 8 %. The preparations were estimated to be 50 % pure, and the results suggest that glycogen synthase kinase 3 is a monomeric enzyme, M , = 54000 .+ 3000.Glycogen synthase kinase 3 phosphorylated three serine residues in glycogen synthase, all contained within nine amino acids in the same tryptic peptide. The rate of formation of the mono-, di-and triphosphorylated derivatives of the peptide showed that the three sites were not phosphorylated either randomly or in a simple sequential manner. The results could be fitted to a model in which either of two serine residues were phosphorylated initially, the phosphorylation of one of these residues being linked to an extremely rapid phosphorylation of the third serine.Glycogen synthase kinase 3 phosphorylated glycogen synthase much more effectively than all other proteins tested. Casein was phosphorylated at a very low rate and with a much higher K,. There was no significant phosphorylation of glycogen phosphorylase, phosphorylase kinase, protein phosphatase inhibitors 1 and 2, 1,-pyruvate kinase, acetyl-CoA carboxylase, ATP-citrate lyase, and histones HI and H2B. The enzyme was also unable to catalyse the inactivation of hydroxymethylglutaryl-CoA reductase.Glycogen synthase kinase 3 underwent an 'autophosphorylation' reaction following incubation with Mg-ATP and up to 4 mol of phosphate could be incorporated per mol of enzyme.The activating factor (FA) of the (Mg-ATP)-dependent protein phosphatase was found to copurify with glycogen synthase kinase 3 throughout the isolation procedure. It also proved impossible to separate the two activities by chromatography on CM-Sephadex or hydroxyapatite, or by gel filtration on Sephadex G-100. The two activities had similar K , values for ATP and GTP. The results suggest that glycogen synthase kinase 3 and factor FA activities reside in the same protein.Neither the catalytic subunit of cyclic-AMP-dependent protein kinase nor phosphorylase kinase were able to activate the (Mg-ATP)-dependent protein phosphatase.The implications of these findings for the regulation of glycogen metabolism, and the mechanism of activation of the (Mg-ATP)-dependent protein phosphatase are discussed.Glycogen synthase can be phosphorylated and inactivated in vitlv by at least three different protein kinases; cyclic-AMPdependent protein kinase, phosphorylase kinase and an enzyme termed glycogen synthase kinase 3 111. The activity of glycogen synthase kinase 3 is unaffected by cyclic AMP, cyclic GMP, calcium ions, calmodulin or the specific protein inhibitor of cyclic-AMP-dependent protein kinase [I]. It has been partially purified from rabbit skeletal muscle and shown to phosphorylate three serine residues on gl...

Debranching enzyme was purified 150-fold from rabbit skeletal muscle by a three-step procedure which utilised ammonium sulphate precipitation, ion-exchange chromatography on DEAE-cellulose and "hydrophobic" chromatography on Sepharose-NH(CH,),NH,. The preparation was completed within three days, and 200 mg enzyme was isolated from 1000 g muscle, which represented an overall yield of 60%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation coefficient, szo, w, was 8.1 S. The amino acid composition was determined, and the absorption coefficient, A;;;, measured refractometrically was 17.5.The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 166000 and this value was supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride (155 000). The molecular weight of the native enzyme measured by high-speed sedimentation equilibrium was 164000, showing that the debranching enzyme is a monomeric protein at the concentrations which exist in muscle (0.7 mgiml). The results indicate that the two different enzyme activities which are associated with debranching enzyme, 1,4-glucan-4-glycosyltransferase and amylo-l,6-glucosidase, reside on the same polypeptide chain.Protein-glycogen particles isolated from skeletal muscle showed seven major protein-staining components by polyacrylamide gel electrophoresis, one of which was identified as debranching enzyme. Four of the other components were the a and subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthetase.A new titrimetric assay for debranching enzyme was developed; it was used to demonstrate that the maximum potential activity of debranching enzyme is only 5 -10

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