L-type glycogen synthase. Tissue distribution and electrophoretic mobility

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.     

The inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

The activity of glycogen synthase phosphatase in rat liver stems from the co-operation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.     

Comparative characterization of human and rat liver glycogen synthase.

Glycogen synthase from human liver was studied, and its properties were compared with those of rat liver glycogen synthase. The rat and human liver glycogen synthases were similar in their pH profile, in their kinetic constants for the substrate UDP-glucose and the activator glucose 6-phosphate, and in their elution profiles from Q-Sepharose. The apparent molecular weight of the human synthase subunit was 80,000-85,000 by gel electrophoresis, which is similar to that of the rat enzyme. In addition, antibodies to rat liver glycogen synthase recognized human liver glycogen synthase, indicating that the enzymes of these two species share antigenic determinants. However, there were significant differences between the two enzymes. In particular, the total activity of the human enzyme was higher than that of the rat. The human enzyme, purified to near homogeneity, had a specific activity of 40 U/mg protein compared with 20 U/mg protein for the rat enzyme. The active forms of the rat enzyme had greater thermal stability than those of the human enzyme, but the human enzyme was more stable on storage in various buffers. Last, amino acid analysis indicated differences between the enzymes of the two species. Since glycogen synthase is an important enzyme in liver glycogen synthesis, the characterization of this enzyme in the human will help provide insight regarding human liver glycogen synthesis.     

Phosphorylation of rabbit liver glycogen synthase by multiple protein kinases

Purified rabbit liver glycogen synthase was found to be a substrate for six different protein kinases: (i) cyclic AMP-dependent protein kinase, (ii) two Ca2+-stimulated protein kinases, phosphorylase kinase (from muscle) and a calmodulin-dependent glycogen synthase kinase, and (iii) three members of a Ca2+ and cyclic nucleotide independent class, PC0.7, FA/GSK-3, and casein kinase-1. Greatest inactivation accompanied phosphorylation by cyclic AMP-dependent protein kinase (to 0.5-0.7 phosphate/subunit, +/- glucose-6-P activity ratio reduced from approximately 1 to 0.6) or FA/GSK-3 (to approximately 1 phosphate/subunit, activity ratio, 0.46). Phosphorylation by the combination FA/GSK-3 plus PC0.7 was synergistic, and more extensive inactivation was achieved. The phosphorylation reactions just described caused significant reductions in the Vmax of the glycogen synthase with little effect on the S0.5 (substrate concentration corresponding to Vmax/2). Phosphorylase kinase achieved a lesser inactivation, to an activity ratio of 0.75 at 0.6 phosphate/subunit. PC0.7 acting alone, casein kinase-1, and the calmodulin-dependent protein kinase did not cause inactivation of liver glycogen synthase with the conditions used. Analysis of CNBr fragments of phosphorylated glycogen synthase indicated that the phosphate was distributed primarily between two polypeptides, with apparent Mr = 12,300 (CB-I) and 16,000-17,000 (CB-II). PC0.7 and casein kinase-1 displayed a decided specificity for CB-II, and the calmodulin-dependent protein kinase was specific for CB-I. The other protein kinases were able, to some extent, to introduce phosphate into both CB-I and CB-II. Studies using limited proteolysis indicated that CB-II was located at a terminal region of the subunit. CB-I contains a minimum of one phosphorylation site and CB-II at least three sites. Liver glycogen synthase is therefore potentially subject to the same type of multisite regulation as skeletal muscle glycogen synthase although the muscle and liver enzymes display significant differences in both structural and kinetic properties.     

Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III glycogen storage disease.

Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.     

Interaction between glycogenin and glycogen synthase

Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, glycogenin-1, and nebulin. The interaction with glycogenin was found to be mediated by the region of glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH2- and COOH-terminal phosphorylation sites are not involved in the interaction. The core segment of glycogen synthase from Glu21 to Gly503 does not bind COOH-terminal fragment of glycogenin. However, this region of glycogen synthase binds full-length glycogenin indicating that glycogenin contains at least one additional interacting site for glycogen synthase besides the COOH-terminus. We demonstrate that the COOH-terminal fragment of glycogenin can be used as an effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver.     

Purification and properties of glycogen synthase I from bovine polymorphonuclear leucocytes

Glycogen synthase I was purified from bovine polymorphonuclear leucocytes (PMNs) by a procedure involving concanavalin A-Sepharose affinity chromatography. The purified glycogen-bound glycogen synthase I had a specific activity of 9.83 U/mg protein and the glycogen free enzyme 21 U/mg protein. Molecular ratio of the native enzyme and the subunit were 340 K and 85 K respectively. After phosphorylation by the catalytic subunit of cAMP-dependent protein kinase the phosphorylated sites were studied using high-performance liquid chromatography (HPLC) of the tryptic 32P-peptides. The enzyme was phosphorylated at three different sites with retention times identical to site 1a, site 1b, and site 2 from rabbit skeletal muscle glycogen synthase.     

Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.     

Rabbit liver glycogen synthase kinases. Characterization of a protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin

A rabbit liver protein kinase (PC0.7), able to phosphorylate glycogen synthase and phosvitin, has been extensively purified. The enzyme had apparent Mr = 170,000-190,000 as judged by gel filtration and was associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000). Two other polypeptides, Mr = 38,000 and Mr = 35,000, were also detected. Treatment with trypsin led to an enzyme composed only of polypeptides of Mr = 35,000 and Mr = 25,000. The beta-polypeptide underwent autophosphorylation when incubated with Mg2+ and ATP or GTP. The protein kinase was effective in utilizing both ATP and GTP as the phosphoryl donor (apparent Km values 5-11 microM and 9-19 microM, respectively). The enzyme phosphorylated phosvitin, casein, and glycogen synthase but not histone or phosphorylase and was inhibited by heparin. Phosphorylation of glycogen synthase proceeded to approximately 0.5 phosphate/subunit with little inactivation of the glycogen synthase. The phosphorylation occurred predominantly in a 21,000-dalton CNBr fragment of glycogen synthase that had been previously shown to reside toward the COOH terminus of the molecule. The liver PC0.7 appeared very similar to an analogous enzyme isolated from rabbit muscle (DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962). The present work, therefore, provides a point of contact between the Ca2+ and cyclic nucleotide-independent glycogen synthase kinases of rabbit liver and muscle.     

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had M_r 250 000, 7.2S and the peak A enzyme M_r 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl₂. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K _m ^app 4.2 m for muscle glycogen synthease I and K _m ^app 45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca²⁺, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - DTT dithiothreitol - EGTA ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Subcellular localization of glycogen synthase with monoclonal antibodies

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.     

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P-labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB-2 phosphorylation was increased by all three hormones while CB-1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2+-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase II (PC0.7) and FA/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK-3, is involved or phosphatase activity is regulated, or both.     

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).     

Kinetic properties of glycogen synthase from skeletal muscle after phosphorylation by glycogen synthase kinase 4

Glycogen synthase I (EC 2.4.1.11) from rat and from rabbit skeletal muscle was phosphorylated in vitro by glycogen synthase kinase 4 (EC 2.7.1.37) to the extent of 0.8 phosphates/subunit. For both phosphorylated enzymes, the activity ratio (activity without glucose 6-P divided by activity with 8 mM glucose 6-P) was 0.8 when determined with low concentrations of glycogen synthase and/or short incubation times. However, the activity ratio was 0.5 with high enzyme concentrations and longer incubation times. It was found that the lower activity ratios result largely from UDP inhibition of activity measured in the absence of glucose 6-P. Inhibition by UDP was much less pronounced for glycogen synthase I, indicating that a major consequence of phosphorylation by glycogen synthase kinase 4 is an increased sensitivity to UDP inhibition.     

Primary structure of rabbit skeletal muscle glycogen synthase deduced from cDNA clones

The complete amino acid sequence of rabbit skeletal muscle glycogen synthase was deduced from cDNA clones with a composite length of 3317 bp. An mRNA of 3.6 kb was identified by Northern blot analysis of rabbit skeletal muscle RNA. The mRNA coded for a protein of 734 residues with a molecular weight of 83,480. The deduced NH2-terminal and COOH-terminal sequences corresponded to those reported for the purified protein, indicating the absence of any proteolytic processing. At the nucleotide level, the 5' untranslated and coding regions were 79 and 90% identical for rabbit and human muscle glycogen synthases, whereas the 3' untranslated regions were significantly less similar. The enzymes had 97% amino acid sequence identity. Interestingly, the NH2 and COOH termini of rabbit and human muscle glycogen synthase, the regions of phosphorylation, showed the greatest sequence variation (15 of 19 mismatches and two insertion/deletion events), which may indicate different evolutionary constraints in the regulatory and catalytic regions of the molecule.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

Immunologic identification of a glycogen synthase 93,000-dalton subunit from rat heart and liver

A polyclonal sheep antibody to rat heart glycogen synthase has been used for immunoblot analysis and immunoprecipitation of both rat heart and liver synthase. The purified antibody completely inhibits glycogen synthase activity in rat heart preparations and specifically blots to a 93-kDa band in the 10,000 X g supernatants of both heart and liver homogenates. Immunoprecipitation of in vitro translation products from rat heart or liver poly(A+) RNA yields a unique band with a molecular mass of 93 kDa. Thus the subunit molecular mass of active glycogen synthase in rat heart is 93 kDa. In rat liver at least one form of glycogen synthase also appears to have a molecular mass of 93 kDa. Protocols used to purify rat liver synthase yield a subunit of 80-87 kDa, which retains activity, but which is no longer recognized by the antibody. This suggests that 1) a specific antigenic sequence has been proteolytically removed from the NH2 or COOH terminus of the protein, or 2) that limited proteolysis has led to a conformational change in the enzyme such that the antibody binding site is no longer recognized. Either or both of these possibilities represent a significant alteration in the enzyme due to proteolysis. In vitro studies using synthase preparations having molecular masses less than 93 kDa must be interpreted with caution due to possible structural changes which occur during purification which may alter the regulation or covalent modification of synthase.     

Hormonal regulation of hepatic glycogen synthase phosphatase

Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of glycogen synthase phosphatase. Expected changes occurred in cAMP, cAMP-dependent protein kinase, glycogen synthase, and glycogen phosphorylase. The effect of glucagon on synthase phosphatase was partially reversed by simultaneous addition of insulin (4 x 10(-8) M), an effect paralleled by a decrease in cAMP. Addition of arginine vasopressin (10 milliunits/ml) resulted in a similar inactivation of synthase phosphatase and activation of phosphorylase, but independent of any changes in cAMP or its kinase. Phosphorylase phosphatase activity was unaffected by any of these hormones. Synthase phosphatase activity, measured as the ability of a crude homogenate to catalyze the conversion of purified rat liver synthase D to the I form, was no longer inhibited by glucagon or vasopressin when phosphorylase antiserum was added to the phosphatase assay mixture in sufficient quantity to inhibit 90-95% of the phosphorylase a activity. These data support the following conclusions: 1) hepatic glycogen synthase phosphatase activity is acutely modulated by hormones, 2) hepatic glycogen synthase phosphatase and phosphorylase phosphatase are regulated differently, 3) the hormone-mediated changes in synthase phosphatase cannot be explained by an alteration of the synthase D molecule affecting its behavior as a substrate, and 4) glycogen synthase phosphatase activity is at least partially controlled by the level of phosphorylase a.     

Phosphorylation of glycogen synthase by the Ca2+- and phospholipid-activated protein kinase (protein kinase C)

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been found to phosphorylate and inactivate glycogen synthase. With muscle glycogen synthase as a substrate, the reaction was stimulated by Ca2+ and by phosphatidylserine. The tumor-promoting phorbol esters 12-O-tetradecanoyl phorbol 13-acetate was also a positive effector, half-maximal activation occurring at 6 nM. Phosphorylation of glycogen synthase, but not histone, was partially inhibited by glycogen, half-maximally at 0.05 mg/ml, probably via a substrate-directed mechanism. The rate of glycogen synthase phosphorylation was approximately half that for histone; the apparent Km for glycogen synthase was 0.25 mg/ml. Protein kinase C also phosphorylated casein, the preferred substrate among the individual caseins being alpha s1-casein. Glycogen synthase was phosphorylated to greater than 1 phosphate/subunit with an accompanying reduction in the -glucose-6-P/+glucose-6-P activity ratio from 0.9 to 0.5. Phosphate was introduced into serine residues in both the NH2-terminal and COOH-terminal CNBr fragments of the enzyme subunit. The two main tryptic phosphopeptides mapped in correspondence with the peptides that contain site 1a and site 2. Lesser phosphorylation in an unidentified peptide was also observed. Rabbit liver and muscle glycogen synthases were phosphorylated at similar rates by protein kinase C. The above results are compatible with a role for protein kinase C in the regulation of glycogen synthase as was suggested by a recent study of intact hepatocytes.     

Regulation of cardiac glycogen synthase by phosphorylation: catalysis of inactivation by cAMP-dependent and cAMP-independent protein kinases and comparison with the phosphorylation of the skeletal muscle enzyme

Glycogen synthase has been purified from bovine heart to near homogeneity by a procedure including zonal sucrose gradient ultracentrifugation. The purified enzyme had a subunit molecular weight of 88,000 ± 2000, an $\text{I}\text{D}$ ratio of between 0.8 and 1.0, and contained less than 0.1 mol of covalently bound phosphate per mole of subunit. The rates, extent, and sites of phosphorylation of the cardiac enzyme were compared with those of skeletal muscle glycogen synthase as catalyzed by both the cardiac cAMP-dependent and a cardiac cAMP-independent protein kinases. The cardiac glycogen synthase was phosphorylated up to 1 mol of phosphate/mol of subunit by the cAMP-dependent protein kinase, to at least 2 mol of phosphate/mol of subunit by the cAMP-independent protein kinase, and to at least 3 mol of phosphate/mol of subunit with the two protein kinases together. There was a linear correlation between the extent of phosphorylation and conversion of cardiac synthase I to the glucose 6-phosphate-dependent form. This correlation was independent of which kinase(s) catalyzed the phosphorylation. Maximum inactivation occurred at an incorporation of 2 mol of phosphate per subunit. Under equivalent conditions, the rates of phosphorylation of cardiac and skeletal muscle glycogen synthase by the cAMP-dependent protein kinase were identical. In contrast, the cardiac enzyme was phosphorylated at a faster rate by the homologous cardiac cAMP-independent protein kinase than was the skeletal muscle synthase by the latter cardiac protein kinase. Analysis of the sites of phosphorylation of the cardiac and skeletal muscle glycogen synthases by CNBr cleavage and trypsin hydrolysis indicated minor differences in the derived phosphopeptides.