Glycogen Phosphorylase and Synthase Activities of Rumen Ciliates of the Genus Entodinium

Glycogen phosphorylase and synthase activities were detected in the sonic lysate of rumen ciliates of the genus Entodinium. The ciliate phosphorylase had the following properties. The pH optimum was narrow and centered at pH 5.9. The activity was maximum at 30°C; above 40°C a rapid inactivation occurred. The K_m value for glucose-1-phosphate (G-1-P) and for glycogen was 15 mM and 0.069% (w/v), respectively. NaF and ethylenediamine tetraacetic acid had no stimulative effect on the enzyme activity, though adenosine 3′,5′-monophosphate and theophylline activated it. NaHSO₃ inhibited the enzyme activity at a concentration of 1 mM. The inhibition of glucose was noncompetitive for G-1-P. Glycolytic intermediates and nucleotides had a minor effect on phosphorylase activity. Glycogen synthase existed in two forms, glucose-6-phosphate dependent and independent forms: the proportion of the latter form increased with the decrease of reserve polysaccharide levels in the ciliates. Correlations between glycolytic enzyme activities included phosphorylase and synthase activities and reserve polysaccharide contents in the ciliates were determined, and a possible regulatory mechanism of polysaccharide synthesis and degradation was discussed.     

Two Glycogen Synthase Activities Associated with Proteoglycogen in Retina

Glycogen synthase of bovine retina was found associated with the acid-insoluble and acid-soluble proteoglycogen fractions. The synthase associated with the acid-insoluble proteoglycogen precursor showed an 8-fold lower Km for UDP-glucose than the synthase associated with the acid-soluble fraction, and was inhibited by detergent. A short digestion with pronase resulted in conversion of the acid insoluble fraction into acid-soluble. The results lead us to postulate that the acid-insolubility of the proteoglycogen fraction and the association with retina membrane proposed before, is caused by glycogen synthase strongly associated to its polysaccharide moiety. The enlargement of the polysaccharide moiety during proteoglycogen biosynthesis, from glycogenin linked to a few 11 to 12 glucose units to the acid-insoluble proteoglycogen precursor (Mr 470,000) would be carried out, together with the branching enzyme, by the glycogen synthase showing a low Km for UDP-glucose. The glycogen synthase with the highest Km for UDP-glucose would participate in conversion of the precursor into mature acid-soluble proteoglycogen.     

Relationship of Glycogen Synthase and Glycogen Phosphorylase to Protein Phosphatase 2C and cAMP-Dependent Protein Kinase in Liver of Obese Rhesus Monkeys

ORTMEYER HK. Relationship of glycogen synthase and glycogen phosphorylase to protein phosphatase 2C and cAMP-dependent protein kinase in liver of obese rhesus monkeys. The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/ threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determined. Basal enzyme activities and basal substrate concentrations were not significantly different between the three groups of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p<0. 05) and to PP2C activity (p=0. 005) (adjusted R²,53%). GP independent activity was correlated to GS independent activity (p<0. 07) and to PKA fractional activity (p=0. 005) (adjusted R²,64%). PP2C activity was correlated to GS fractional activity (p<0. 0005) and to PP1/2A activity G7<0. 0001) (adjusted R²,83%). PKA fractional activity was correlated to GP total activity (p<0. 0005) and to age (p=0. 001) (adjusted R282%). G6P content was correlated to glycogen content (p<0. 05) and to PP2C activity (p=0. 0005) (adjusted R²,73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.     

Localization of Glycogen Synthase in Brain

Antisera against glycogen synthase from canine brain were prepared and used for investigation of the localization of the enzyme in the brain. Antisera cross-reacted only with the 88-kilodalton protein that is the subunit of brain glycogen synthase. Immunoreactivity of glycogen synthase was universally distributed in all regions of the brain, although hippocampus, cerebral cortex, caudatoputamen, and cerebellar cortex had relatively high immunoreactivity. Light microscopic examination revealed that the immunoreactivity was found in all cell types, such as neurons in several regions, astrocytes, ependymal cells surrounding the ventricle, oligodendrocytes, and epithelial cells of the choroid plexus in the ventricle. Immunoreactive intensity was more prominent in neurons than glial cells. Immunostaining may be a useful tool for investigation of the state of glycogen metabolism under normal and pathological conditions.     

Yeast Glycogen Phosphorylase: Characterization of the Dimeric Form and Its Activation

The purification of yeast glycogen phosphorylase [EC 2.4.1.1] was improved by ethanol precipitation and affinity chromatography on a glycogen-Sepharose column. The purified enzyme gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a subunit molecular mass of 100 kDa. Gel electrophoresis also showed that the major activity of native phosphorylase was ascribed to a dimer of 203 kDa, which was agreed with the value obtained by gel filtration on Sephadex G-200. The yeast phosphorylase showed a high affinity for AMP- Sepharose, whereas the enzyme was specifically inhibited by AMP. This inhibition was competitive with respect to the substrate glucose 1-phosphate and gave a Ki value of 9.3 mm. Activation of the crude extract by phosphorylation with an endogenous phosphorylase kinase indicated that the yeast phosphorylase occurred in a mixture of phosphorylated and non-phosphorylated forms.     

Nuclear Localization of Brain-type Glycogen Phosphorylase in Some Gastrointestinal Carcinoma

Our previous reports have demonstrated frequent and strong expression of glycogen phosphorylase (EC 2.4.1.1) activity mainly in the cytoplasm of gastric carcinoma. Although previous studies have suggested the phosphorylase glyco-syltransferase system to be in the nucleus from enzyme histochemical analyses, intranuclear localization of the phosphorylase has not been fully established. The aims of the present study are to investigate the nuclear localization of glycogen phosphorylase and to identify the isoform of phosphorylase in the nucleus of gastrointestinal carcinoma. The activity of glycogen phosphorylase in carcinoma cells corresponding to the nucleus was demonstrated using enzyme cytochemical analysis. The phosphorylase activity coincided with localization revealed by immunocytochemistry using affinity-purified specific anti-human brain-type glycogen phosphorylase antibody. The isoform expressed in the nuclei of carcinoma cells was identified as bei ng only the brain type according to a polymerase chain reaction-based assay using RNA obtained from gastric carcinoma cells and primers specific to muscle, liver and brain types of glycogen phosphorylase. The intranuclear localization of the brain-type isoform was confirmed by immunoelectron microscopical analyses. Further investigation to examine the nuclear localization in human carcinoma tissue (145 and 25 specimens with gastric and colonic carcinoma respectively) was carried out by immunohistochemistry using specific anti-brain-type antibody. Nuclear immunostaining was observed in seven cases out of 145 gastric carcinoma. The present study is the first to clarify the nuclear localization of glycogen phosphorylase with enzymatic activity in gastrointestinal carcinoma. The isoform of the enzyme expressed in the carcinoma was identified as the brain type. These results warrant further studies on the mechanisms for transporting the large molecule of brain-type glycogen phosphorylase to nuclei and its function in the nucleus of carcinoma cells.     

Glycogen Metabolism in Chlamydia-Infected HeLa-Cells

A difference in glycogen metabolism between two strains of Chlamydia agents was observed which can serve as a taxonomic marker to distinguish C. psittaci from C. trachomatis.     

Modeling K,ATP-Dependent Excitability in Pancreatic Islets

In pancreatic β-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic β-cell excitability reproduces the β-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca²⁺ channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of β-cell excitability and suggests future experiments that will lead to improved characterization of β-cell excitability and the control of insulin secretion.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Modeling K,ATP-Dependent Excitability in Pancreatic Islets

In pancreatic β-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic β-cell excitability reproduces the β-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca²⁺ channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of β-cell excitability and suggests future experiments that will lead to improved characterization of β-cell excitability and the control of insulin secretion.     

Glycogen Metabolism in Aureobasidium Pullulans: A Glycogen Synthetase with Unusual Activation Properties

Abstract

Cultures of filamentous fungi that secrete significant amounts of exopolysaccharides are among the most difficult of fermentation fluids, presenting difficulties in the areas of aeration, agitation, mixing, and control that may in turn impact the physiology of the microorganism in an undesirable manner. The fungus Sclerotium glucanicum, which produces a potentially useful exopolysaccharide known as scleroglucan, illustrates many such difficulties. This review discusses in detail the range of physiological studies on the producing microorganism itself, including those concerning formation of “undesirable” byproducts, principally oxalate, but also, under certain conditions, other TCA cycle acids. In addition, the bioreactor technology in use for production of this type of biopolymer is discussed in relation to the difficulties such fluid types present. The potential of pneumatically agitated reactors for such production is evaluated, and the lack of fundamental studies on such reactors and on the hydrodynamics and mixing behavior of such complex fluids is pointed out.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Metabolic Response to Nonglucidic Nutrient Secretagogues and Enzymatic Activities in Pancreatic Islets of Adult Rats after Neonatal Streptozotocin Administration

In islets from adult rats injected with streptozotocin during the neonatal period, both a nonmetabolized analog of L-leucine and 3-phenylpyruvate augmented ¹⁴CO₂ output from islets either prelabeled with L-[U-¹⁴C]glutamine or exposed to D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose in a manner qualitatively comparable to that found in islets from control rats. The islets of diabetic rats differed, however, from those of control rats by their unresponsiveness to both the L-leucine analog and a high concentration of D-glucose in terms of increasing ³HOH generation from [2-³H]glycerol, an impaired sparing action of the hexose upon ¹⁴CO₂ output from islets prelabeled with [U-¹⁴C]palmitate, and, most importantly, by a decreased rate of D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose oxidation when either incubated at a high concentration of the hexose (16.7 mM) or stimulated by nonglucidic nutrient secretagogues at a low concentration of D-glucose (2.8 mM). In islet homogenates, the activity of glyceraldehyde phosphate dehydrogenase, glutamate decarboxylase, and NADP-malate dehydrogenase was lower in diabetic than control islets. Such was not the case for glutamatealanine transaminase, glutamate-aspartate transaminase, or glutamate dehydrogenase. The neonatal injection of streptozotocin thus affected, in the adult rats, the activity of several islet enzymes. Nevertheless, the metabolic data suggest that an impaired circulation in the glycerol phosphate shuttle, as observed in response to stimulation of the islets by either a high concentration of D-glucose or nonglucidic nutrient secretagogues, represents an essential determinant of the preferential impairment of glucose-induced insulin release in this model of non-insulin-dependent diabetes.     

肌糖原磷酸化酶a,b亚基的杂交

本文用标记在肌糖原磷酸化酶ａ（ＧＰＡ）亚基上的荧光探针ＩＡＥＤＡＮＳ与标记在肌糖原磷酸化酶ｂ（ＧＰＢ）亚基上的ＩＡＦ之间无辐射能量转移;高压力下亚基交换动力学和外源荧光探针荧光偏振等三种方法证实了肌糖原磷酸化酶ａ、ｂ的亚基可相互交换,形成磷酸化酶ａ、ｂ的杂交形式。     

Activation of Glycogen Phosphorylase by Cyclic AMP in Tetrahymena pyriformis1

Glycogen phosphorylase in Tetrahymena pyriformis was activated by a Mg²⁺ ATP-dependent process and this activation was further increased by the addition of cyclic AMP. When the enzyme activity in subcellular fractions was measured, it was largely associated with the glycogen fraction but was no longer activated by ATP and cyclic AMP. Mixing the glycogen fraction and cytosol fraction together restored the effects of ATP and cyclic AMP on phosphorylase activity. These findings suggest that glycogen phosphorylase associated with Tetrahymena glycogen granules may be regulated by cytosolic factor(s) with cyclic AMP.     

Glycogen Metabolism in Bovine Adrenal Medulla

Abstract: Glycogen content was determined both in whole adrenal medullary tissue and in isolated adrenal chromaffin cells, in which it responds to glucose deprivation and restoration. [¹⁴C]glucose incorporation into glycogen in isolated adrenal chromaffin cells is increased by previous glucose deprivation ("fasting"). Total glycogen synthase activities are 452 ± 66 mU/g in whole tissue and 305 ± 108 mU/g in isolated cells. The K _m of glycogen synthase for UDP-glucose is 0.67 mM with 13 m m glucose-6-phosphate and 1 m m without this effector. The in vitro inactivation process of glycogen synthase a has been found to be mainly cyclic AMP-dependent, but it also responds to Ca²⁺. Total glycogen phosphorylase activities are 8.69 ± 1.26 U/g in whole tissue and 2.38 ± 0.30 U/g in isolated cells. The requirements for interconversion in vitro of both glycogen synthase and phosphorylase suggest a system similar to that of other tissues. During incubation of isolated adrenal chromaffin cells with 5 m m -glucose, phosphorylase a activity decreases and synthase a activity increases; these changes are more marked in "fasted" cells. Glycogen content and glycogen synthase and phosphorylase activities are higher in the adrenal medulla than in the brain, suggesting a greater metabolic role of glycogen in the adrenal medulla.