Form Of Glycogen Synthetase Research Articles

Activities of glycogen synthetase and glycogen phosphorylase in the human endometrium: Relative distribution in isolated glands and stroma

The activities of glycogen synthetase and glycogen phosphorylase were studied in endometrial samples obtained from 51 premenopausal women during the menstrual cycle. The total activities of glycogen synthetase and glycogen phosphorylase and the activity of the active form of glycogen phosphorylase increased gradually from the proliferative phase to the secretory phase and reached a maximum during the midsecretory phase, while the activity of the active form of glycogen synthetase increased slightly. In 30 of the 51 women, the relative distribution of glycogen synthetase and glycogen phosphorylase activities in isolated glands and stromal cells was determined following collagenase digestion of the endometrial specimens. The results indicated that the activities of the active form of glycogen synthetase and glycogen phosphorylase in the isolated glands during the secretory phase were more than threefold and twofold, respectively, greater than those present in the isolated stromal cells and that the levels of these enzymes in the glands and stromal cells changed in parallel with those in the undissociated endometrium observed during the menstrual cycle. In addition, histochemical studies revealed the presence of glycogen phosphorylase activity in both the glands and the stromal cells, whereas the glycogen synthetase activity was present only in the glands. These findings suggest that the stromal cells of the human endometrium as well as the glands may play an important role in the nutrition of the implanting blastocyst.

Hepatic glycogen synthetase deficiency not expressed in cultured skin fibroblasts

Cultured skin fibroblasts from two siblings with hepatic glycogen synthetase deficiency and from their parents contained glycogen synthetase activity which was within the range of the controls. The mutation was thus not expressed in fibroblasts, a finding further documenting the presumed existence of genetically different forms of glycogen synthetase.

Glycogen synthetase in the sea mussel Mytilus edulis L.—I. Purification, interconversion and kinetic properties of the I and D forms

1. 1. The two forms, I (active, independent of G6P) and D (inactive, dependent on G6P), of glycogen synthetase have been identified in Mytilus edulis. 2. 2. Incubation of a crude extract of the mantle enzyme at 30°C resulted in conversion of the D to I form due to endogenous phosphatase activity. 3. 3. The mantle enzyme was purified by affinity chromatography on Con A-Sepharose. The K m values for UDPG were 0.32 and 2.8 mM for the D and I forms of the enzyme, respectively. The K a for G6P, of the D form was 0.76 mM. 4. 4. Inorganic phosphate (2–10 mM) strongly inhibited the D form of glycogen synthetase. The inhibition was not reversed by increased concentrations of G6P (10 mM); in contrast, 5 mM G6P reversed the inhibition of the I form by inorganic phosphate.

Amino acid sequences at the two sites on glycogen synthetase phosphorylated by cyclic AMP-dependent protein kinase and their dephosphorylation by protein phosphatase-III

It has been established that glycogen synthetase activity in skeletal muscle can be regulated by two distinct protein kinases. One of these is cyclic AMPdependent protein kinase, while the other is an enzyme termed glycogen synthetase kinase-2 [ 1,2] . The phosphorylation of glycogen synthetase a in vitro by either kinase in the presence of ATP-Mg was found to reach a plateau when about one molecule of phosphate had been incorporated per subunit, and the resulting enzyme species were more dependent on the allosteric activator glucose-6-phosphate for activity. However cyclic AMP dependent protein kinase and glycogen synthetase kinase-2 produced different forms of glycogen synthetase, termed bi and bz respectively. These forms had different activity ratios (defined as the activity in the-absence of glucose-6-phosphate relative to the activity in the presence of this effector) and different activation constants (K,) for glucosedphosphate [2] . Moreover, the addition of both protein kinases resulted in the incorporation of almost two molecules of phosphate per subunit and the conversion of glycogen synthetase to a form, termed bl,2, which was almost completely inactive in the absence of glucosed-phosphate [2] . These results indicated that

The effect of enzymes upon metabolism, storage, and release of carbohydrates in normal and abnormal endometria.

This paper presents preliminary data concerning the relationship of various components of glandular epithelium and effect of enzymes on metabolism, storage, and release of certain substances in normal and abnormal endometria. Activity of these endometrial enzymes has been compared between two groups: 252 patients with normal menstrual histories and 156 patients, all over the age of 40, with abnormal uterine bleeding. Material was obtained by endometrial biopsy or curettage. In the pathologic classification of the group of 156, 30 patients had secretory endometria, 88 patients had endometria classified as proliferative, 24 were classified as endometrial hyperplasia, and 14 were classified as adenocarcinoma. All tissue was studied by histologic, histochemical, and biochemical methods. Glycogen synthetase activity caused synthesis of glucose to glycogen, increasing in amount until midcycle, when glycogen phosphorylase activity caused the breakdown to glucose during the regressive stage of endometrial activity. This normal cyclic activity did not occur in the abnormal endometria, where activity of both enzymes continued at low constant tempo. Only the I form of glycogen synthetase increased as the tissue became more hyperplastic. With the constant glycogen content and the increased activity of both the TPN isocitric dehydrogenase and glucose-6-phosphate dehydrogenase in the hyperplastic and cancerous endometria, tissue energy was created, resulting in abnormal cell proliferation. These altered biochemical and cellular activities may be the basis for malignant cell growth.

Factors Influencing Pituitary Glycogen Metabolism and Gonadotropic Hormone Release. I. Luteinizing Hormone-Releasing Hormone

The way in which luteinizing hormone (LH) and follicle stimulating h ormone (FSH) are related to pituitary glycogen metabolism is investigated by measuring pituitary glycogen content and the activity of glycogen synthetase and glycogen phosphorylase in LH-releasing hormone (LH-RH) treated rat pituitaries. The presence of LH-RH (15 ng/flask) resulted in a significant (p less than .05) release of LH and FSH after 4 hours of incubation. Pituitary LH content was also raised (p less than .05). 3 ng of LH-RH increased pituitary cyclic adenosine monophosphate (AMP) content by 475% in 10 minutes of incubation. Glycogen phosphorylase was converted to its active form with 1-10 ng LH-RH per flask; this glycogenolytic activity occurred within 5 minutes (p less than .01) and was also induced by cyclic AMP in the presence of theophylline. The forms of glycogen synthetase did not appear to be affected by LH-RH or cyclic AMP. It is concluded that pituitary glycogen may be an energy source for release of gonadotropins via LH-RH.

Multiple molecular forms of phosphoprotein phosphatase from rabbit skeletal muscle

1. 1. Phosphohistone phosphatase activity of rabbit skeletal muscle was separated into two fractions (phosphatase I and phosphatase II) by Sephadex G-150 chromatography. The molecular weights estimated using a Sepharose 6B column for phosphatase I and phosphatase II were about 300 000 and 150 000, respectively. 2. 2. Phosphatase I was active in the absence of divalent metal ions and was not inhibited by EDTA or EGTA. However, the activity of phosphatase II was almost totally dependent on Mn 2+ or Co 2+ with K m of 0.8 and 0.5 mM, respectively. Freezing of phosphatase I in the presence of 200 mM mercaptoethanol resulted in a change of the molecular size to that of phosphatase II with the disappearance of substrate ( P-histone)-inhibition property, which was observed in phosphatase I at a concentration higher than 10 μM of phosphohistone (based on the alkali-labile 32P content). 3. 3. Glycogen synthetase-D phosphatase activity, which converts the D form of glycogen synthetase (UDPglucose:glycogen 4-α-glucosyltransferase, EC 2.4.1.11) to the I form, was also found in the two fractions. Synthetase-D phosphatase activity of phosphatase I was less dependent on Mn 2+ than that of phosphatase II, which was essentially inactive without the metal ion. 4. 4. Existence of the two types of phosphohistone phosphatase was shown in crude extract of rat liver, kidney, brain, heart muscle and skeletal muscle.

Glycogen synthetase and phosphorylase in developing chick glycogen body.

AbstractGlycogen synthetase and phosphorylase activities were quantitatively determined for the first time in glycogen body tissue from late embryonic and neonatal chicks. For comparative purposes, these enzyme activities were examined also in skeletal muscle and liver from pre‐ and post‐hatched chicks. The present data show that the forms of glycogen synthetase which exist in the chick glycogen body are similar to those found in both liver and muscle, while glycogen body phosphorylases more closely resemble those forms characteristic of skeletal muscle.

Yeast Glycogen Synthetase in the Glucose 6-Phosphate-dependent Form: I. PURIFICATION AND PROPERTIES

Abstract The glucose-6-P-dependent form of glycogen synthetase has been purified about 5,000-fold from yeast extracts. The enzyme was separated from the glucose-6-P-independent form by DEAE-cellulose chromatography and from other proteins by a two-step agarose chromatography, using complex formation with glycogen to change the elution properties of the synthetase. The purified material shows a single band on disc gel electrophoresis and possesses a molecular weight of 300,000, as measured by sedimentation equilibrium. Acrylamide electrophoresis in the presence of sodium dodecyl sulfate yields a major band, molecular weight about 77,000, and a minor band of variable intensity, with molecular weight about 71,000. The faster band is believed to be a proteolysis degradation product of the slow band. Thus, the native enzyme would consist of four subunits of equal molecular weight. The enzyme was labeled with radioactive phosphate by conversion into the glucose-6-P-independent form followed by incubation with [γ-32P]ATP. The amount of radioactivity incorporated corresponds to 1.4 phosphate groups per subunit. Incubation with a muscle protein phosphatase led to a liberation of radioactivity and a parallel increase in glucose-6-P-independent synthetase activity. Lineweaver-Burk plots with the purified enzyme, using either UDP-glucose or glycogen as the variable substrate, showed a biphasic curve, as previously observed with cruder enzymes. Increasing concentrations of glucose-6-P led to a gradual elimination of the line corresponding to a higher Km value.

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.

Yeast glycogen synthetase in the glucose 6-phosphate-independent form: A case of cold lability without major changes in molecular size

The form of yeast glycogen synthetase independent of glucose 6-phosphate is reversibly inactivated by cold in the absence of substrates. Addition of glycogen or of a high concentration of glycerol completely prevents the inactivation, whereas glucose 6-phosphate, uridine diphosphate glucose and (NH 4) 2SO 4 afford partial protection. Sedimentation coefficients of 11.2 S at 30 °C and 10.1 S at 0 °C were calculated from centrifugation in sucrose density gradients. This small difference appears to be due to a change in conformation rather than to dissociation of the enzyme into subunits in the cold. The glucose 6-phosphate-dependent form of glycogen synthetase does not display cold sensitivity.

Glycogen Synthetase-D Phosphatase: I. SOME NEW PROPERTIES OF THE PARTIALLY PURIFIED ENZYME FROM RABBIT SKELETAL MUSCLE

Abstract Purification of the phospho-protein phosphatase of skeletal muscle which promotes the conversion of the phospho- (D or b) form to the dephospho- (I or a) form of glycogen synthetase (UDPG:glycogen α-4-glucosyltransferase, EC 2.4.I.II) was aided by the use of buffers which include manganese chloride. The Mn2+ (5 mm) appeared to stabilize the enzyme and to insure greater recovery from DEAE-cellulose chromatography than reported previously. The enzyme was purified more than 1,000-fold from the 78,000 x g supernatant of rabbit muscle homogenate. It was essentially free of phosphorylase and synthetase. Phosphatase activity was measured by two methods: (a) as the rate of conversion of glycogen synthetase-D to the I form (D to I conversion) and (b) as the rate of orthophosphate release from 32P-labeled synthetase-D (32Pi release). Inhibition of the phosphatase reaction (more than 50%) was found in the presence of F- (10 mm), Na2SO3 (1 mm), and Pi or PPi (0.2 mm). More than a 2-fold increase in phosphatase activity was found in the presence of divalent metal cations: Mn2+ g Ca2+ g Mg2+ (half-maximal concentration was 0.6 to 1.2 mm). Glucose-6-P (up to 1 mm) had no effect on the rate of 32Pi release, but increased about 2-fold the rate of D to I conversion (at 0.1 mm), as did galactose-6-P and glucosamine-6-P to a lesser extent. A glycogen concentration of 0.5 to 1.5 mg per ml was found to be optimal compared to the slower reaction rates found with greater or lesser concentrations. A histone phosphatase activity found in this preparation, measured using 32P-labeled histone phosphate as substrate, had properties similar to and was copurified with glycogen synthetase-D phosphatase. However, the above carbohydrate-type effectors of synthetase and synthetase-D phosphatase reactions were without effect on the rate of histone dephosphorylation. These findings suggest that these two phosphatase activities may reside in the same enzyme protein.

Glycogen synthetase of rat skeletal muscle and hepatomas and its comparison with the enzyme of rat liver

1. 1.Properties of glycogen synthetase of rat skeletal muscle and two ascites hepatomas (AH-66F and AH-130) were studied in comparison with rat liver enzyme. The standard assay employed the Tris-maleate buffer of pH 7.4 and 5 mM UDP-glucose. 2. 2.When fresh extracts of muscle and hepatomas were incubated at 30 °C, glycogen synthetase underwent an activation due to the D to I conversion, but unlike the activation observed in the liver enzyme, only the activity measured without glucose 6-phosphate was increased with time. 3. 3.The D and I forms of glycogen synthetase were prepared from muscle, AH-66F and AH-130. The pH-activity curves of the D forms from these tissues differed from that of liver enzyme in that in the presence of glucose 6-phosphate, the activity measured at pH 7.4 was as high as that at pH 8.6. 4. 4.The apparent Michaelis constants for UDP-glucose of the D forms of muscle, AH-66F and AH-130 determined in the presence of glucose 6-phosphate were 0.50, 0.22 and 0.15 mM, respectively. These values are much lower than that reported for the D form of liver (8 mM). 5. 5.Glycogen synthetases of muscle and liver are thus mutually different proteins. There was a close resemblance between the muscle and hepatoma enzymes, suggesting that hepatocarcinogenesis may have resulted in the replacement of liver-specific synthetase by some other type, possibly of muscle.

Effects of adenosine 3',5'-monophosphate and adenosine 5'-monophosphate on glycogen degradation and synthesis in Dictyostelium discoideum.

Data are presented demonstrating that the presence in vivo of adenosine 3',5'-monophosphate (3',5'-AMP) causes a rapid depletion of glycogen storage material in the cellular slime mold. The effect of adenosine 5'-monophosphate (5'-AMP) is twofold, stimulating both glycogen degradation and synthesis. In pseudoplasmodia, cell-free extracts appear to contain at least two species of glycogen phosphorylase, one of which is severely inhibited by glucose-1-phosphate and another which is only partially inhibited by this hexose-phosphate. In some cases, 5'-AMP partially overcomes the inhibition by glucose-1-phosphate. Data presented here also indicate the existence of two forms of glycogen synthetase, the total activity of which does not change during 10 hr of differentiation from aggregation to culmination. During this period there is a quantitative conversion of glucose-6-phosphate-independent enzyme activity to glucose-6-phosphate-dependent activity. It is suggested that one effect of 3',5'-AMP is closely related to enzymatic processes involved in the rapid conversion of glycogen to cell wall material and other end products accumulating during sorocarp construction.

Identification of a third form of glycogen synthetase in rat chloroma tumors

Identification of a third form of glycogen synthetase in rat chloroma tumors

Histochemical differentiation of two forms of glycogen synthetase.

Glycogen synthetase exists in two forms; one form is dependent on glucose 6-phosphate while the other is not. Variations in the levels of activities of the two forms of the enzyme were observed histochemically in active and inactive eccrine sweat glands from eight rhesus monkeys. The clear cells of active sweat glands showed less glucose 6-phosphate-independent glycogen synthetase activity than the clear cells of inactive sweat glands.

Regulation of glycogen metabolism in the adrenal gland: I. Kinetic and regulatory properties of glycogen synthetase

The properties of glycogen synthetase from rat adrenal that may he related to the regulation of glycogenesis in this gland were investigated. It was found that the enzyme sediments at 60,000 g and that it exists at least in two forms, one independent (I-form) and the other dependent on glucose-6-P for activity. The latter is the predominant species in the adrenal of normal rats. ATP, ADP, and P i inhibited to different extents both forms of glycogen synthetase, and this inhibition could be reversed by glucose-6-P, much more efficiently with I-glycogen synthetase. The differential effect of these metabolites on the two glycogen-synthetase forms, and the kinetic constants determined for UDP-glucose and glucose-6-P, suggest that the glycogenetic regulation in the adrenal gland might take place by an interconversion process and/or by the eventual fluctuation in the concentration of these metabolites. The activity of the glycogen synthetase obtained directly, and measured in the absence of glucose-6-P, has kinetic parameters and heat stability different from those of the I-form (obtained in vitro), suggesting that both activities might belong to different forms of enzyme.

The effect of starvation on glycogen synthetase activity in the heart and skeletal muscle of the rat

Eighteen, 48 and 96 hour periods of starvation lead to a progressive, marked decline in both total and independent, active “I” form of glycogen synthetase in the rat heart. The relative percent of “I” remains unchanged until the 96th hour at which time it, too, decreases. Skeletal muscle synthetase remains relatively unaffected during starvation.

Effect of antagonistic albumin on insulin-stimulated intracellular metabolic pathways.

Previous data on incorporation of glucose C-14 into glycogen by the rat diaphragm had suggested that this synthetic pathway was unaffected by the albumin-associated insulin antagonist. To test the hypothesis that only the transport functions of insulin were blocked by this antagonist, studies on two insulin-stimulated intracellular pathways independent of transport were carried out. The increase in tissue levels of the I form of glycogen synthetase seen with insulin was not affected by antagonistic albumin (human fraction V). The D form of glycogen synthetase was also unaffected. However, insulin-stimulated adenine-8-C-14 incorporation into RNA was completely abolished by this albumin. A nonantagonistic albumin (bovine fraction V) by itself increased RNA synthesis indicating that the inhibition of insulin-stimulated RNA synthesis was not a general property of the albumin molecule. The physical-chemical and metabolic characteristics of the albumin-associated antagonist are summarized and are considered to be incompatible with the postulated physiological role for this insulin antagonist.