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.     

Methadone inhibition of glycogen synthase and phosphorylase in rat skeletal muscle

The activities of glycogen synthase (I and total) and phosphorylase ($\text{a}$ and total) in crude extracts of isolated extensor digitorum longus and soleus muscles of the rat incubated $\text{in vitro}$ in the absence or presence of methadone were very low. Addition of glycogen during homogenization increased the activities of both enzymes in control muscles. Even at optimal concentrations of glycogen, however, the activities of both enzymes from methadone-treated muscles were significantly lower than their activities in control muscles. The activity of phosphoglucomutase was not altered by incubation with methadone or by homogenization with glycogen. It is suggested that the addition of optimal amounts of glycogen during extraction of the enzymes enhances the extractability of glycogen synthase and increases the activity of phosphorylase by some other mechanism and that these processes are interfered with when the muscles are pretreated with methadone.     

Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies

Isozyme-specific antibodies were raised against peptides from the low-homology regions of the sequences of rat glycogen phosphorylase BB and MM isozymes by immunization of rabbits and guinea pigs. Immunocytochemical double-labelling experiments on frozen sections of rat nervous tissues were performed to investigate the isozyme localization pattern. Astrocytes throughout the brain and spinal cord expressed both isozymes in perfect co-localization. Ependymal cells only expressed the BB isozyme. Most neurones were not immunoreactive. The rare neurones that contained glycogen phosphorylase only expressed the BB isozyme. Nearly all of these neurones formed part of the afferent somatosensory system. These findings stress the general importance of glycogen in neural energy metabolism and indicate a special role for the glycogen phosphorylase BB isozyme in neurones in the somatosensory system.     

The nucleotide sequence of rat liver glycogen phosphorylase cDNA.

The nucleotide sequence of a cDNA coding for rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the cDNA are sufficient to encode the total protein as determined by comparison with the liver type of glycogen phosphorylase of man. Human and rat liver glycogen phosphorylase showed 86% homology at the DNA level whereas the deduced amino acid sequence has 93.5% identity.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Isolation and study of a liver alpha-amylase. Comparison with glycogen phosphorylase activity

An oligomaltosaccharide-forming amylase has been observed in mice liver crude homogenate. This enzyme has been isolated by binding to amylose. Some of its functional parameters have been studied and compared with those of glycogen phosphorylase demonstrating that amylase activity is not due to a glycogen phosphorylase isoenzyme. It has been further observed that amylase needs Ca2+ of Mg+2 and Cl- for its activity.     

Adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscle with increased glycogen content

The effect of glycogen content on the activation of glycogen phosphorylase during adrenaline stimulation was investigated in soleus muscles from Wistar rats. Furthermore, adrenergic activation of glycogen phosphorylase in the slow-twitch oxidative soleus muscle was compared to the fast-twitch glycolytic epitrochlearis muscle. The glycogen content was 96.4 +/- 4.4 mmol (kg dw)(-1) in soleus muscles. Three hours of incubation with 10 mU/ml of insulin (and 5.5 mM glucose) increased the glycogen content to 182.2+/-5.9 mmol (kg dw)(-1) which is similar to that of epitrochlearis muscles (175.7+/-6.9 mmol (kg dw)(-1)). Total phosphorylase activity in soleus was independent of glycogen content. Adrenaline (10(-6) M) transformed about 20% and 35% (P < 0.01) of glycogen phosphorylase to the a form in soleus with normal and high glycogen content, respectively. In epitrochlearis, adrenaline stimulation transformed about 80% of glycogen phosphorylase to the a form. Glycogen synthase activation was reduced to low level in soleus muscles with both normal and high glycogen content. In conclusion, adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscles with increased glycogen content. Glycogen phosphorylase activation during adrenaline stimulation was much higher in epitrochlearis than in soleus muscles with a similar content of glycogen.     

The interaction of muscle glycogen phosphorylase b with glycogen

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen was studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64 mumol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of Mr 5.5 X 10(6). Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0 degrees C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0 degrees C, whereas the bimolecular rate constant changes slightly and is about 10(8) M-1 X S-1. These data point to the possibility of diffusional control of the complex formation.     

Complete cDNA sequence for rabbit muscle glycogen phosphorylase

The cDNA for the nearly full-length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position-1, suggesting that the removal of only a single methionine residue precedes the amino-terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat.

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Amylin activates glycogen phosphorylase in the isolated soleus muscle of the rat

The pancreatic beta-cell hormone amylin acts in isolated rat skeletal muscle to decrease insulin-stimulated incorporation of glucose into glycogen. It also increases blood levels of lactate and glucose in fasted rats in vivo. However, it remained uncertain whether amylin exerts direct effects to stimulate muscle glycogenolysis. We now report that amylin caused a dose-dependent increase in activity of muscle glycogen phosphorylase in isolated rat soleus muscle by stimulating phosphorylase a. Insulin inhibited amylin-stimulated activation of phosphorylase. Effects of amylin to stimulate muscle glycogenolysis are consistent with observed effects of amylin in vivo and could be a major mechanism whereby amylin modulates carbohydrate metabolism.     

Quantitation of muscle glycogen phosphorylase mRNA and enzyme amounts in adult rat tissues

Mammalian glycogen phosphorylases comprise a family of isozymes that are expressed selectively in a variety of cell types. As an initial step towards understanding the molecular processes that regulate the differential expression of the phosphorylase family, we have begun a quantitative examination of isozyme expression in vivo. In this paper, we report quantitative estimates of the amounts of the muscle (M) isozyme and its mRNA in adult rat tissues. Quantitative estimates of the amount of M-phosphorylase were obtained by an analysis involving electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose filters and sequential treatment with M-isozyme specific antibody and radioactively- labeled protein A. M-phosphorylase mRNA amounts were determined by an analysis involving transfer of RNA from agarose gels to nitrocellulose filters and subsequent hybridization with radioactively labelled rat M-phosphorylase cDNA. These studies indicate that M-phosphorylase is present in all tissues tested with the possible exception of liver. These are skeletal muscle, heart, brain, stomach, lung, kidney, spleen and testis. Quantitation of M-phosphorylase amounts indicate that there is a wide spectrum of variation (over 1000-fold range) in the relative amounts of the M-isozymes in these tissues. Relative mRNA levels parallel isozyme levels indicating that the major control of expression of this isozyme is governed by mRNA accumulation.     

Kinetics of the reaction between muscle glycogen phosphorylase B and glycogen

Kinetics of glycogen binding by glycogen phosphorylase b has been studied by stopped flow and temperature jump methods. This reaction is followed by increase in light scattering whose amplitude depends upon the enzyme binding sites concentration of glycogen particles occupied by the enzyme. It has been shown that the complex formation has the first order with respect to enzyme and glycogen concentrations. Relaxation kinetics is compatible with proposed bimolecular reaction scheme. Microscopic rate constants of the forward and reverse reactions of glycogen binding by glycogen phosphorylase b are determined in temperature range from 12,7 to 30 degrees C. The possibility of diffusional control of the binding rate is discussed.     

Enzyme kinetics of muscle glycogen phosphorylase b

Interest in the kinetics of glycogen phosphorylase has recently been renewed by the hypothesis of a glycogen shunt and by the potential of altering phosphorylase to treat type II diabetes. The wealth of data from studies of this enzyme in vitro and the need for a mathematical representation for use in the study of metabolic control systems make this enzyme an ideal subject for a mathematical model. We applied a two-part approach to the analysis of the kinetics of glycogen phosphorylase b (GPb). First, a continuous state model of enzyme-ligand interactions supported the view that two phosphates and four ATP or AMP molecules can bind to the enzyme, a result that agrees with spectroscopic and crystallographic studies. Second, using minimum error estimates from continuous state model fits to published data (that agreed well with reported error), we used a discrete state model of internal molecular events to show that GPb exists in three discrete states (two of which are inactive) and that state transitions are concerted. The results also show that under certain concentrations of substrate and effector, ATP can activate the enzyme, while under other conditions, it can competetively inhibit or noncompetitively inhibit the enzyme. This result is unexpected but is consistent with spectroscopic, crystallographic, and kinetic experiments and can explain several previously unexplained phenomena regarding GPb activity in vivo and in vitro.