Affinity glycogen synthetase to crosslinked glycogen.

The affinity of yeast glycogen synthetase to glycogen modified by crosslinking has been studied under various experimental conditions. It was found that the higher the degree of crosslinking, the lower the affinity of glycogen synthetase to glycogen. The amount of glycogen synthetase adsorbed from the solution depends on the amount of crosslinked glycogen added and is inversely proportional to the concentration of the soluble glycogen. The stability of the complex formed between yeast glycogen synthetase and the crosslinked glycogen was found to be maximal at neutral pH range. The presence of glucose 6-phosphate, uridine 5′-di-phosphate, and uridine 5′-diphosphate glucose enhanced the stability of the complex.     

Histochemical studies on glycogen synthetase activity by autoradiographic method

Summary Glycogen synthetase (uridine diphosphate glucose-glycogen glucosyl transferase) was studied in different organs by a histoautoradiographic method and by usual staining methods. This activity was found to be present in muscles and liver of different animals. Human skin also showed some activity. Human liver and myocardium showed the highest activity.In the present study, it was found that the glucose-6-phosphate dependent form (D-form) of the glycogen synthetase predominates over the glucose-6-phosphate independent form (I-form) in all the organs except hamster liver where the I-form predominates.Addition of calcium chloride in the incubation medium, to prevent phosphorolytic breakdown of the newly synthesized glycogen, does not improve the reaction. No glucose is incorporated into glycogen from ¹⁴C-glucose-6-phosphate of the incubation medium for glycogen synthetase. Fixation in absolute alcohol at –20° is recommended for tissues where cytolysis is caused by the incubation medium.     

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., Endocrinology88, 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.     

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.     

Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during the growth (myxamoebal) phase

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 that are grown in axenic medium containing 86mm-glucose have seven times the glycogen content of the same myxamoebae grown in the same medium but lacking added carbohydrate. 2. During the transition from the exponential to the stationary phase of growth in axenic medium containing glucose myxamoebae preferentially synthesize glycogen and can have as much as three times the glycogen content during the stationary phase as they have during the exponential phase of growth. 3. The rate of glycogen degradation by myxamoebae is, under all conditions of growth, small compared with the rate of glycogen accumulation and the changes in glycogen content thus reflect altered rates of glycogen synthesis. 4. There is no correlation between the rate of glycogen synthesis by myxamoebae and the glycogen synthetase content of the myxamoebae. 5. The activity of glycogen synthetase of D. discoideum is inhibited by a physiological concentration of ATP and this inhibition is overcome by glucose 6-phosphate. Both effects are especially marked at physiological concentrations of UDP-glucose. 6. The rate of glycogen accumulation by myxamoebae growing exponentially in axenic media can be satisfactorily accounted for in terms of the known intracellular concentrations of glucose 6-phosphate, UDP-glucose and glycogen synthetase. The rate-limiting factors controlling glycogen synthesis by the myxamoebae are apparently the substrate (UDP-glucose) and effector (glucose 6-phosphate and ATP) concentrations rather than the amount of the enzyme.     

Uridine and cytidine nucleotide synthesis in renal hypertrophy: Biochemical differences in response to the growth stimulus of diabetes and unilateral nephrectomy

The effects of unilateral nephrectomy (UN) and streptozotocin (STZ) diabetes on the activities of enzymes involved in uridine and cytidine synthesis in early renal growth (3–14 days after stimulus to growth) have been compared. Measurements were also made of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) and of glucose 6-phosphate (G6P), UDP-glucose, and glycogen, in relation to phosphoribosyl pyrophosphate, ribonucleotide, and complex carbohydrate formation. There were striking differences in the activities of CTP synthetase, G6PDH, and 6PGDH in the two conditions, with a three-fold increase in all three enzymes at 3 and 5 days and a two-fold increase above basal values at 14 days of STZ diabetes. The UN group showed no significant change in CTP synthetase at any stage and the activity of G6PDH and 6PGDH only kept pace with renal growth. Changes in routes of uridine synthesis were less marked, with a more rapid rise in carbamoyl-phosphate synthetase (glutamine) and a lesser response of dihydroorotate dehydrogenase in the UN relative to the STZ-diabetic groups. The enzymes of complex II and of uracil phosphoribosyltransferase showed essentially similar patterns during renal hypertrophy in UN and STZ diabetes. The parallel increase in CTP synthetase, G6PDH, and 6PGDH in the kidney in diabetes, also known to increase in growth situations in hepatomas and in renal tumors, is discussed in relation to hormone signals involved in renal growth. The importance of the concentration of CTP, and thus of CTP synthetase, in the CTP-cytidyltransferase reaction, an enzyme with a high K_m for CTP, makes the present observation of the striking increase in CTP synthetase in STZ diabetes of particular interest in relation to phosphatidylcholine formation and hormone signal transduction.     

Kinetics of the D and I glycogen synthetase forms and their interconversion, in the sea scallop Pecten maximus.

In extracts from the adductor muscle of the shell-fish, Pecten maximus, glycogen synthetase (EC.2.4.1.11) was found. The enzyme occurs predominantly as D form (glucose-6-P dependent for activity). An I form (G-6-P independent) was also present. Kinetics of glycogen synthetase showed that the Ka for G-6-P in the D form was 10 fold higher than in the I form. Both forms of glycogen synthetase were interconverted through reactions catalyzed by phosphatase and kinase enzymes respectively. Glucose-6-P and Mg+2 must be present to stabilize glycogen synthetase and to activate the synthetase D phosphatase, found in the 90,000 X g protein-glycogen complex. The conversion of synthetase D to I was inhibited by F-, glycogen, ATP and UTP. When F- was present the effect of G-6-P on synthetase and phosphatase suggested that conversion involved the existence of more than a single glycogen synthetase phosphatase enzyme. ATP and Mg+2 were necessary for the conversion of synthetase I to D, and the conversion was stimulated by cAMP.     

Carbohydrate nutrition before, during, and after exercise

The role of dietary carbohydrates (CHO) in the resynthesis of muscle and liver glycogen after prolonged, exhaustive exercise has been clearly demonstrated. The mechanisms responsible for optimal glycogen storage are linked to the activation of glycogen synthetase by depletion of glycogen and the subsequent intake of CHO. Although diets rich in CHO may increase the muscle glycogen stores and enhance endurance exercise performance when consumed in the days before the activity, they also increase the rate of CHO oxidation and the use of muscle glycogen. When consumed in the last hour before exercise, the insulin stimulated-uptake of glucose from blood often results in hypoglycemia, greater dependence on muscle glycogen, and an earlier onset of exhaustion than when no CHO is fed. Ingesting CHO during exercise appears to be of minimal value to performance except in events lasting 2 h or longer. The form of CHO (i.e., glucose, fructose, sucrose) ingested may produce different blood glucose and insulin responses, but the rate of muscle glycogen resynthesis is about the same regardless of the structure.     

Chemical and Biochemical Changes in Subcellular Fractions of Guinea Pig Liver During Infection with Coxiella burneti

Livers of uninfected guinea pigs and of guinea pigs infected with Coxiella burneti were fractionated into smooth endoplasmic reticulum, rough endoplasmic reticulum (RER), pellet, and cell sap fractions. The ribonucleic acid (RNA) and protein of each fraction were determined, and the phosphorylase, glucose-6-phosphatase, and glucosyl transferase (glycogen synthetase) activities of each fraction were measured. Decreased RNA, protein, and enzyme activities were found in the RER and pellet fractions of infected livers, with the greatest differences in the RER. The evidence indicates a solubilization of the phosphorylase and synthetase, with the enzymes moving from the RER and glycogen-containing pellet fraction to the cell sap. The data suggest the RER as a target during Q fever.     

Glycogen and glycogen enzymes in the liver and striated muscle of rats under altered thyroid states

Changes induced in liver and striated muscle glycogen and glycogen enzymes (glycogen synthetase, glycogen phosphorylase and alpha-amylase) by hypothyroidism and hyperthyroidism in rats have been determined. There were no changes in liver glycogen synthetase, phosphorylase and amylase activities in the hypothyroid group. Hyperthyroid rats showed lower liver glycogen synthetase, phosphorylase a and amylase activities. In muscle, hypothyroid rats had lower phosphorylase activity. In the hyperthyroid group glycogen synthetase was increased.--The results presented do not completely agree with the glycogen levels found in both tissues studied, and they are obviously more related to other factors such as glucose availability. It can be concluded that under the conditions studied, the glycogen enzyme levels could not alone explain the variations of glycogen levels.     

Hormonal control of endometrial glycogen metabolism in the Macaca arctoides

Endometrial biopsies obtained throughout the menstrual cycle of the Macaca arctoides show the glycogen content paralleling the serum progesterone fluctuations which occur during the menstrual cycle. Secretory phase samples contained a three-fold higher concentration of glycogen when compared to follicular phase tissue. Changes in the activity levels of the glycogen metabolizing enzymes, glycogen phosphorylase and glycogen synthetase, during various stages of the menstrual cycle are in accord with the concept that the post-ovulatory increase in endometrial metabolism is a function of progesterone influence on this tissue. Endometrial glycogen synthetase activity remains low during the early proliferative phase of the cycle and becomes significantly elevated (two-to three-fold) during the early secretory phase of the cycle. Glycogen phosphorylase shows a similar cyclicity later in the luteal phase, reaching maximal activity between the seventeenth to nineteenth day of the cycle and remaining elevated through the twenty-sixth day of the cycle. The coincident nature of the rise in peripheral progesterone to increases in uterine glycogen metabolism suggest that progesterone may be the prime modulator of uterine endometrial metabolism during the post-ovulatory phase.     

Regulation of glycogen synthetase and phosphorylase phosphatase activities in rat adipose tissue.

Incubation of a rat adipose tissue homogenate causes a time and temperature dependent activation of glycogen synthetase (UDP glucose:glycogen 4-alpha-glucosyltransferase) and simultaneous inactivation of phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Activation of glycogen synthetase at 15 and 23 degrees C was preceded by a lag period. The duration of the lag period could not be correlated with significant changes in phosphorylase activity. Addition of glucose and methylxanthines caused an increase in the rates of glycogen synthetase activation and phosphorylase inactivation. The effect on glycogen synthetase activation was mainly on the linear phase. Addition of AMP inhibited phosphorylase inactivation and accelerated glycogen synthetase activation. Addition of muscle phosphorylase alpha caused a prolongation of the lag period which lasted until phosphorylase alpha activity had decreased to the level originally present in the preparation. It is concluded that in adipose tissue activation of glycogen synthetase is not dependent on prior inactivation of phosphorylase and that other factors should be looked for to explain the lag period preceding glycogen synthetase activation.     

Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli.

A mutant of Escherichia coli harboring a temperature-labile phosphoribosylpyrophosphate (PRPP) synthetase was characterized. Despite the lack of a detectable PRPP pool or PRPP synthetase activity at 40 degrees C, the strain was fully viable at this temperature as long as guanosine, uridine, histidine, tryptophan, and nicotinamide mononucleotide were all added to the growth medium. Viability of the strain was dependent upon mutations in genes of the nucleoside salvage pathways that improved the utilization of exogenous nucleosides. The properties of the strain are those expected of a PRPP-less strain and suggest that PRPP synthetase is dispensable for E. coli.     

Changes in glycogen metabolism in chick embryo liver in relation to the formation of glycosomes]

In the chick embryo liver the portion of granular glycogen increases from 15 to 90% of the total content during the period from the 8th till the 14th days of developments. The activity of glycogen synthetase (KF 2.4.1.11) localized in the fraction of granular glycogen increases from 40 to 90% of the total activity in the 18 days old embryo. The activity of phosphorylase (KF 2.4.1.1) is detected in the granular glycogen of the liver only on the 12th day of development (10% of the total activity) and increase up to 80% on the 19th day of development. The maximal activation of glycogen synthetase and phosphorylase is noted after the glycosomes of formation in the developing embryoliver. A suggestion is put forward to the effect that the process of glycosome formation is a factor of the control of glycogen synthetase and phosphorylase activity.     

Biosynthesis of Glycogen and Starch in Cryptococcus laurentii

Cells of Cryptococcus laurentii, when grown in liquid culture on 2% glucose close to neutral pH, showed glycogen granules throughout the cytoplasm. Glycogen levels of C. laurentii cells reached maximal levels just before onset of stationary phase. Concomitantly, a sharp rise in total and specific activity of glycogen synthetase was observed. Conversely, glycogen phosphorylase reached its highest specific activity approximately 3 hr after the glycogen peaked and remained high until most of the endogenous glycogen was utilized. Uridine diphosphoglucose pyrophosphorylase activity was always an order of magnitude higher than glycogen synthetase during log phase, but fell off rapidly after the cells reached stationary growth. Kinetic properties of the glycogen synthetase showed that the enzyme is always activated by glucose-6-phosphate, although the degree of activation by glucose-6-phosphate was found to be somewhat variable. The accelerated uptake of glucose commencing with the onset of stationary phase is explained by the rapid formation of extracellular acidic polysaccharide, which continues as long as there is glucose in the medium. In cells grown at pH 3.4, where no detectable extracellular acidic polysaccharide was formed, glucose uptake drastically declined when the cells reached stationary phase. These cells also contained glycogen-like granules in the cytoplasm. The evidence presented indicates that these granules are in fact glycogen, and that its structure does not resemble that of the starch excreted by cells grown at acidic pH.     

Mechanisms of Pathogenesis in Listeria monocytogenes Infection III. Carbohydrate Metabolism

Several enzymes and metabolites concerned with carbohydrate metabolism were examined in mice infected with Listeria monocytogenes. Liver glycogen and glucose decreased parallel to severity of infection. The concentration of glucose in the blood fell to abnormally low levels with a hypoglycemia being most evident at 72 hr. There was a significant decrease in the activity of hepatic uridine diphosphate glucose-glycogen transglucosylase. This decrease in enzymatic activity correlated with the rate of glycogen depletion. Phosphorylase activity declined in a similar fashion, contraindicating enhanced glycogenolysis as the mechanism responsible for glycogen depletion. Although glucose-6-phosphatase decreased throughout the infection period, it did not appear to be the major metabolic defect causing hypoglycemia in Listeria-infected mice. Further distortion of carbohydrate metabolism was indicated by findings of increased levels of pyruvate and lactate in the blood of infected animals.     

Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: I. Kinetic Aspects

Treatment of pea stem segments with indoleacetic acid (IAA) causes within 1 hour a 2- to 4-fold increase in activity of particulate uridine diphosphoglucose-dependent beta-glucan synthetase obtainable from the tissue. The IAA effect is observable in tissue from all parts of the elongation zone of the pea stem, and also in older tissue that is not capable of a cell enlargement response to IAA. A large increase in activity is caused by IAA only if synthetase activity in the isolated tissue has first been allowed to fall substantially below the intact plant level, and only if sucrose is supplied along with IAA. Treatment of tissue with sucrose alone after a period of sugar starvation causes a transient rise of synthetase activity. The decline in synthetase activity in absence of IAA, the rise caused by IAA, and the transient rise caused by sucrose are all strongly temperature-dependent. IAA and sucrose do not affect the activity of isolated synthetase particles. Synthetase activity in vivo is sensitive to as low as 0.1 mum IAA and is increased by IAA analogues that are active as auxins on elongation but not by nonauxin analogues. Activity begins to rise 10 to 15 minutes after exposure to IAA, which places this among the most rapid enzyme effects of a plant growth regulator heretofore demonstrated, and among the most rapid known metabolic effects of auxins. The effect is seen also with polysaccharide synthetase activity using uridine diphosphate-galactose or uridine diphosphate-xylose as substrates, and to a lesser extent with guanosine diphosphoglucose-dependent glucan synthetase activity. Glucan synthetase from IAA-treated tissue appears to have a higher affinity for uridine diphosphate-glucose than the control.     

Glutamine synthetase and fructose-1, 6-diphosphatase activity in the putamen of control and Huntington's disease brain post mortem

There is a linear negative correlation between the activities of glutamine synthetase and fructose-1, 6-diphosphatase in normal Human putamen autopsy samples, and also in the Huntington's disease putamen. However, glutamine synthetase activity is reduced in choreic brain samples, while fructose-1, 6-diphosphatase activity is normal. The ratio of fructose-1, 6-diphosphatase to glutamine synthetase is therefore increased in Huntington's disease. The products of the two reactions, glutamine and fructose-6-phosphate, are the starting substrates for glycolipid and glycoprotein biosynthesis, via the glutamine:fructose-6-phosphate aminotransferase catalysed formation of glucoseamine-6-phosphate. The alternative metabolic route of fructose-6-phosphate leads to glycogen. The availability of glutamine, and the activity of glutamine synthetase may control fructose-6-phosphate metabolism, and the increased ratio of fructose-1,6-diphosphatase to glutamine synthetase in Huntington's disease may explain the accumulation of glycogen, and the reduction in ganglioside levels reported in this state.     

The effects of estrogen and progesterone on glycogen and the enzymes involved in its metabolism in the cat uterus

In the endometrium of the spay cat, estradiol treatment causes a drastic reduction in glycogen content. The initiation of simultaneous progesterone treatment causes a replenishment of glycogen with the levels surpassing those observed in the spayed animal. The effect of the hormones on glycogen synthetase I follows the same pattern as the changes in glycogen content, while the effect on phosphorylase a is the mirror image. The total content of glycogen synthetase and phosphorylase do not show appreciable changes with the same hormone treatment. The possible reproductive role of these changes in glycogen content are discussed.     

Modifications of the activities of key enzymes and intracellular levels of cyclic nucleotides, in correlation with the glyogen deposition in a cultured hepatoma cell line.

Glycogen accumulation in growing cultures of ZHC cells (originally derived from the Zajdela ascitic hepatoma) is accompanied by an increase in glycogen synthetase (E.C. 2.4.1.11) and phosphorylase (E.C. 2.4.1.1) activities. Essentially the synthetase b and the phosphorylase a are involved in this process. The glycogen accumulation in ZHC cells us preceeded by a noticeable peak of cAMP, whereas cGMP rises early after replating and then decreases simultaneously with the growth rate. The present results suggest that these cultured hepatoma cells undergo throughout every passage an induction process involved in glycogen synthesis storage. Since the original ascites cells growing in vivo (which lack glycogen) and the cultured ZHC cells exhibit similar glycogen synthetase and phosphorylase activities, the resurgence of the glycogenic function (Staedel and Beck, 1978) in the in vitro cultureed cells does not seem related to a change in these two enzymes. By contrast, the high cyclic nucleotide levels in the cultured cells, as compared to those in the ascites cells, offer a possible explanation.