Glycogen Synthetase Phosphatase Research Articles

Glycogen metabolism of the human endometrium.

The glycogen concentration and the activities of glycogen synthetase and phosphorylase were assayed in endometrial tissue obtained during curettage. Tissues were obtained during different phases of the menstrual cycle from women investigated because of menometrorrhagia or sterility. The glycogen concentration was less than 2 mg/g wet wt in specimens from days 1-15 and increased 10-fold during the early secretory phase. The total activities of glycogen synthetase and phosphorylase were increased 1.5- and 2-fold, respectively, during the secretory phase compared to those during the first half of the cycle. The activities of the active forms (a form) of these enzymes did not show significant changes during the menstrual cycle. The largest difference between the proliferative and secretory phases was in the activity of glycogen synthetase phosphatase, which was virtually inactive in tissues obtained on days 1-15 and increased almost 20-fold during the secretory part of the cycle. In tissues obtained from cases of sterility, the activities of glycogen synthetase and phosphorylase were significantly reduced only during the early secretory phase. Glycogen concentration throughout the menstrual cycle and enzyme activities during the midsecretory and premenstrual phase were similar in cases of sterility and other pathologies.

Effect of Dietary Energy on Hepatic Glycogen Metabolism in the Turkey Hen

An experiment was conducted with turkey hens to investigate the effect of substituting 30% of the carbohydrate calories with corn oil, 1,3-butanediol, or glycerol. Birds fed additional corn oil had the lowest liver glycogen concentration. Corn oil increased phosphorylase a, total phosphorylase, and glycogen synthetase I in comparison to the controls. Also, additional corn oil resulted in the highest specific activity of glucose-6-phosphatase. Dietary glycerol caused the highest concentration of liver glycogen. Glycerol increased glycogen synthetase I, but had little effect upon total activity in comparison to butanediol in the diet. Both butanediol and glycerol gave similar phosphorylase a activity, but butanediol increased total activity. The fat-fed and control-fed hens regulated hepatic glycogen concentration through phosphorylase, while glycerol and butanediol-fed hens regulated glycogen through glycogen synthetase.In vitro activation of glycogen synthetase I was deficient in hens fed additional corn oil, indicating a lack of glycogen synthetase phosphatase activity. The order of activation (glycerol > butanediol > control > corn oil) corresponds to the rank of glycogen concentrations.

The Rigulation of Glycogen Metabolism

Inhibitor-1 from rabbit skeletal muscle was phosphorylated by protein kinase dependent on adenosine 3' :5'-monophosphate (cyclic AMP), but not by phosphorylase kinase or by glycogen synthetase kinase-2. Protein phosphatase-III, isolated and stored in the presence of manganese ions to keep it stable, was in a form which catalysed a rapid dephosphorylation and inactivation of inhibitor-1. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 0.7 micron, V(rel) = 40] were comparable to those for the dephosphorylation of phosphorylase kinase [Km =1.1 micron, V (rel) = 62] and phosphorylase [Km = 5.0 micron, V (rel) = 100]. The dephosphorylation of inhibitor -1 was inhibited by inhibitor-2, indicating that it was catalysed by protein phosphatase-III, and not by another enzyme that might be contaminating the preparation. When protein phosphatase-III was diluted into buffers containing excess EDTA, it lost activity initially, but after 90 min, the activity reached a plateau that remained stable for at least 20h. The initial loss in activity varied with the substrate that was tested; it was 20-30% with phosphorylase a, 50-60% with phosphorylase kinase and greater than or equal to 95% with inhibitor-1. This form of protein phosphatase-III was inhibited by inhibitor-1 in a noncompetitive manner, and the Ki for inhibitor-1 was 1.6 +/- 0.3 nM. The phosphorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthetase phosphatase activities of protein phosphatase-III were inhibited in an identical manner by inhibitor-1. This result emphasizes the potential importance of inhibitor-1 in the regulation of glycogen metabolism, since it can influence the state of phosphorylation of three different enzymes. The formation of the inactive complex between inhibitor-1 and protein phosphatase-III was reversed by incubation with trypsin (which destroyed inhibitor-1, but not protein phosphatase-III) or by dilution of the inactive complex. Kinetic studies, using the form of protein phosphatase-III which dephosphorylated inhibitor-1 very rapidly, demonstrated three unusual features of the system: (a) inhibitor-1 was still as powerful and inhibitor of the dephosphorylation of phosphorylase a and phosphorylase kinase a even under conditions where it was being rapidly dephosphorylated; (b) inhibitor-1 was not an inhibitor of its own dephosphorylation; (c) phosphorylase a did not effect the rate of dephosphorylation of inhibitor-1 even when it was present in a 50-fold molar excess over inhibitor-1. The result of these three properties is that inhibitor-1 is preferentially dephosphorylated by protein phosphatase-III even in the presence of a large excess of other phosphoprotein substrates. Inhibitor-1 was also dephosphorylated by protein phosphatase-II. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 2.8 micron, V (rel) = 200] and the alpha-subunit of phosphorylase kinase [Km = 3.7 micron, V (rel) = 100]were comparable...

Effect of limited proteolysis on activity and phosphorylation of rabbit muscle glycogen synthetase

Purified rabbit skeletal muscle glycogen synthetase, in both the glucose-6-phosphate (P)-dependent (phosphorylated) and the glucose-6-P-independent (dephosphorylated) forms, was subjected to limited proteolysis by trypsin. Both forms could be degraded from their original subunit molecular weight of 85,000 to 76,000 and subsequently to 68,000, as determined with acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Degradation of the glucose-6-P-dependent form of the enzyme resulted in essentially no change in the activity when measured either in the presence or in the absence of glucose-6-P. Degradation of the glucose-6-P-independent form was associated with a progressive increase in glucose-6-P dependency. Phosphorylation of the glucose-6-P-independent form with the adenosine 3′,5′-monophosphate-dependent protein kinase and subsequent digestion of the 32P-labeled enzyme showed that the phosphate group was retained on these subunits. The protein kinase phosphorylated both the original subunit with molecular weight 85,000 and the partially digested subunit with molecular weight 76,000. Upon further digestion of the enzyme into a form having a subunit molecular weight of 68,000, the enzyme was unable to accept a phosphate group from ATP. By contrast with the phosphorylation reaction, the dephosphorylation reaction catalyzed by partially purified glycogen synthetase phosphatase is not stringent in terms of structural integrity of the synthetase. The phosphatase dephosphorylated the glucose-6-P-dependent form of glycogen synthetase equally well at various degrees of degradation.

Glycogen synthetase kinase 2 (GSK 2); The identification of a new protein kinase in skeletal muscle

Glycogen synthetase (EC 2.4.1.11) is regulated through a phosphorylation-dephosphorylation mechanism [l] . The dephosphorylated I-form is fully active in both the presence and absence of glucosedphosphate, and can be converted to the phosphorylated D-form, which has an absolute requirement for glucose&phosphate, by incubation with magnesium ions, ATP and the cyclic AMP dependent protein kinase which also activates phosphorylase kinase [2,3] The proportion of glycogen synthetase D in skeletal muscle is increased by the administration of adrenalin [4] . This presumably acts through the elevation in cyclic AMP levels [ 5,6] and activation of cyclic AMP dependent protein kinase [7] which have been shown to occur in vivo in response to the hormone. The reconversion of glycogen synthetase D to I is under the control of insulin [8] , but it appears to be independent of the effect of this hormone on glucose transport into muscle [4] . It is also unlikely that the effect is explainable in terms of an inactivation of cyclic AMP dependent protein kinase resulting from a decreased availability of cyclic AMP, because cyclic AMP levels in muscle not only do not decrease in the presence of insulin, but even show a slight rise [9]. Theoretically, the elevation of synthetase I levels by insulin could be achieved in one of three different ways. Insulin could increase the activity of glycogen synthetase phosphatase, decrease the activity of cyclic AMP dependent protein kinase, or change the conformation of glycogen synthetase itself, rendering it a better substrate for the phosphatase or a less effective substrate for the kinase. Since an investigation of these possibilities required the use of both the D and I forms

Early changes of protein-kinase (glycogen-synthetase kinase) in denervated muscle

After denervation, muscle glycogen decreases in simultaneously with glycogen synthetase I activity (the physiologically active form of this enzyme). The present experiment was performed to determine the changes, if any, in the enzymes that convert glycogen synthetase from the inactive D form to the active I (glycogen synthetase phosphatase) as well as the opposite reaction (glycogen synthetase kinase or protein kinase). Enzyme activities were assayed by testing the capability of muscle extracts to cause conversion of purified glycogen synthetase D (in the case of phosphatase determinations) or of purified glycogen synthetase I (kinase assays) in the presence of the required cofactors and activators. No changes of glycogensynthetase phosphatase were observed. However, there was a marked increase of both the cAMP independent and the total kinase activities 1 day after denervation. This fact can account for the changes in glycogen synthetase I levels and in glycogen metabolism after denervation. Also, it might be relevant to understand the early changes in RNA and protein metabolism after denervation, owing to the supposed regulatory effect of protein kinase on nuclear activity.

Studies on glycogen metabolism in normal and diabetic rat heart in vivo.

A study has been made on the key enzymes involved in synthesis and breakdown of glycogen and the major intermediates in glycogen synthesis in isolated normal and streptozotocin diabetic rat heart. A significant loss of hexokinase isoenzyme type I and type II was observed in the soluble fraction of hearts from starved and diabetic rats.No significant change was observed in total and active form of glycogen synthetase and phosphorylase in diabetic rat heart, but glycogen synthetase phosphatase activity in vitro was found to be considerably less. Treatment with insulin restored the synthetase phosphatase in vitro activity, and also significantly increased synthetase I in vivo.Tissue content of the major intermediates on the glycogen synthetic pathway, glucose 6-phosphate, glucose 1-phosphate, and UDP-glucose, was found to be significantly higher in diabetic rat heart than in normal. Insulin (4 I.U./kg for 30 min) was able to normalize the high values of tissue content of the glycogen precursors found in diabetic heart. The decreased glycogen synthesis in diabetic rat heart seems to be mainly due to the decreased activity of glycogen synthetase phosphatase.

Regulation of glycogen metabolism in the adrenal gland: IV. The effect of insulin on glycogen synthetase, phosphorylase, and related metabolites

Previous studies have indicated that the glycogen content of adrenal glands of fasted rats can be depleted by insulin per se (Bindstein, E., Piras, R., and Piras, M. M., Endocrinology 88, 223, 1971). In order to establish the mechanism of action of this hormone in the adrenal gland, the effect of insulin has been now investigated on glycogen synthetase (UDP-glucose: α-1,4 glucan α-4-glueosyl-transferase, EC 2.4.1.11), glycogen phosphorylase (α-1,4 glucan: orthophosphate glucosyl-transferase, EC 2.4.1.1) and metabolites related to these enzymes. Approximately 40% of total adrenal glycogen phosphorylase of fasted rats is in the active form, which increases to 75% 1 hr after insulin treatment (75 mU/100 g body wt). This conversion occurs without apparent large changes of 3′-5′ cyclic AMP. Concomitantly with the enzymatic change, the levels of glucose-6- P, UDP-glucose and P i suffer alterations which favor an increased phosphorolytic activity during the first hour of insulin treatment. Glycogen synthetase, which did not change during this period, is converted to the glucose-6- P independent form during the 2–3 hr of treatment. This conversion is preceded by an increased glycogen synthetase phosphatase activity, which seems to follow an inverse relationship with the glycogen level. The results obtained suggest that the effect of insulin on the adrenal gland of fasted rats is glycogenolytic, that is, opposite to that described for this hormone in other normal tissues. The glycogen depletion, on the other hand, seems to set in motion the mechanism for glycogen synthetase activation, with the subsequent glycogen resynthesis.

The control of liver glycogen synthetase phosphatase by phosphorylase.

The activation of glycogen synthetase in a liver gel filtrate and the latency by which it is normally preceded have been studied in conditions where the level of phosphorylase a was greatly modified by the addition of either the pure enzyme or of antibodies directed against it. In the presence of liver or muscle phorphorylase a, glycogen synthetase phosphatase was nearly inactive; phosphorylase b had only a slight effect. Antibodies, obtained in the rabbit against dog liver phosphorylase, strongly depressed the activity of phosphorylase a in a mouse liver preparation; they simultaneously allowed an immediated activation of glycogen synthetase.These results indicate that the lag period that precedes that activation of glycogen synthetase in a liver filtrate is the time required for the conversion of phosphorylase a into b by phosphorylase phosphatase. The previously reported shortening of the lag period by glucose, caffeine and nicotinamide or by a tratment with glucocorticoids is entirely explained by the faster inactivation of phosphorylase in the same conditions. The elongation of the lag period by glycogen and its supporession by AMP and magnesium remained however to be explained. It has been shown that the dose of glycogen that prolongs the lag period also inhibits phosphorylase posphatase, wherease AMP and magnesium counteract the inhibition of synthetase phosphatase by phosphorylase a.

The effect of glucose and of a treatment by glucocorticoids on the activation in vitro of liver glycogen synthetase.

The removal of glucose, AMP and other small molecules from mouse liver extract by filtration through Sephadex G-25 has allowed us to demonstrate that the activation in vitro of glycogen synthetase is much more rapidly attained in the presence of glucose and also when the animals have received prednisolone 3 h before sacrifice. These effects are the result of a shortening of the lag period that precedes the activation of the synthetase; they are conveniently studied in filtrates that have been enriched with sulfate or phosphate ions. Like glucose, caffeine and nicotinamide shorten the lag period and are without action when this period is over; α-particulate glycogen has the opposite effect. No lag period is observed in the presence of AMP, particularly when associated with magnesium acetate. When a liver Sephadex filtrate is incubated in the absence of salts, glycogen synthetase b is converted into a partially active form that is rapidly transformed into synthetase a upon the addition of salt. These results support the hypothesis that glycogen synthetase phosphatase is initially present in the liver filtrate as a b enzyme, active only in the presence of AMP and magnesium; this b form is converted into its active, a, homologue through the action of a synthetase phosphatase activating enzyme, which is more active after a treatment with glucocorticoids, is stimulated by glucose, caffeine and nicotinamide and inhibited by glycogen, AMP and fluoride.

Control of rat liver glycogen synthetase and phosphorylase activities by glucose

In the perfused rat liver, hyperglycemia causes a rapid inactivation of phosphorylase and a conversion of glycogen synthetase from a glucose-6- P dependent form to a glucose-6- P independent form. This effect is similar in the presence or in the absence of added insulin, but is absent following adrenalectomy and fasting when glycogen synthetase phosphatase activity is not detectable. Under conditions where phosphatase activity is low, glucose increases its activity. The effect of glucose occurs in the absence of detectable alterations in tissue concentration of adenosine 3′,5′-monophosphate, the primary intracellular regulator mediating hormonal control of the activity of these enzymes. Circulating glucose levels also directly modify the effects of glucagon on the activities of these enzymes supporting the possibility that glucose levels may be important in moderating the control of liver glycogen metabolism in vivo .