Enzyme activity during the metabolism of glycogen. I. Demonstration of phosphorylases in the sensory cells of the tuberous organ of Gnathonemus petersii (Mormyridae). Histochemical and ultrastructural methods.

Phosphorylase activities were investigated by histochemical and ultrastructural procedures in the electroreceptive sensory cells of the tuberous organ of Gnathonemus petersii. Ater incubation in G1P, G1P activated by AMP (Takeuchi and Kuriaki medium) or in G1P activated by ATP+MgSO4 (Guha and Wegman medium) newly formed polysaccharides were analysed with the iodine and P.A.S. reactions under light microscopy and, under electron microscopy, with the periodic acid thiocarbohydrazide (TCH) silver proteinate (PATAg reaction, Thiery), The newly formed polysaccharides proved the presence of glycogen phosphorylase (2.4.1.1) activities and of their branching enzymes (2.4.1.18). When G1P was activated by ATP+MgSO4, they appeared as glycogen particles with the same constitution as native glycogen. After incubation in G1P and in G1P activated by AMP they appeared as glycogen and polyglucose filaments too. In the latter case they were high concentrated. The results show that the phosphorylases are principally present in this sensory cell in their inactive form.     

Glycogen metabolism in novikoff ascites-hepatoma cells

A study of the enzymes of the glycogen pathway in Novikoff ascites hepatoma shows that glycogen synthetase has the lowest activity and that the tumour contains no high-K(m) soluble glucokinase. However, incubation of tumour cells with metabolizable sugars in vitro, or intraperitoneal administration of glucose into the tumour-bearing rat, results in glycogen accumulation by the tumour cells. Glycogen synthesis in the tumour is supported by aerobically produced ATP but is decreased anaerobically and by uncouplers of oxidative phosphorylation. Absence of P(i) from the incubation medium increases glycogen synthesis and decreases glycolysis. The optimum temperature for glycogen synthesis is 37 degrees . The capacity of the intact tumour cell to degrade deposited glycogen is low, but is accelerated by 2,4-dinitrophenol. Tumour homogenates prepared after osmotic shock do not incorporate [(14)C]glucose into glycogen. The glucose moiety of glucose 1-phosphate and of UDP-glucose is incorporated into glycogen by the homogenates and the incorporation of glucose 1-phosphate is greatly enhanced by AMP. Glucose 6-phosphate is a poor precursor of glycogen in the homogenate system, probably because it inhibits activation of phosphorylase b by AMP.     

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.     

Effects of added adenine nucleotides on renal carbohydrate metabolism

1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or P(i) or the formation of complexes with Mg(2+) ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30-50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C(3) phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of alpha-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes.     

Some regulatory properties of glycogen phosphorylase from Streptococcus salivarius

Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) of Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K_m for AMP was 0.25 mm and was decreased to 0.125 mm in the presence of NaF. The K_m for NaF was 0.49 m and was decreased to 0.40 m in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 mm AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K_m values for glucose-1-P and glycogen in the direction of synthesis, but increased the V of the enzyme.When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P₂ had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P₂ and ATP, both in the presence and absence of AMP + NaF.     

Probing into the function of the gene product responsible for glycogen storage disease type Ib

This study aimed at directly assessing glucose 6-phosphate (G6P) transport by intact rat liver microsomes. Tracer uptake from labeled G6P occurred with T(1/2) values that proved insensitive to unlabeled G6P or 100 microM vanadate, and could not be activated over background levels by intravesicular phosphate in the complete absence of G6P hydrolysis. [(32)P]Phosphate efflux was similarly unaffected by G6P or phosphate in the incubation medium. We conclude that the gene product responsible for glycogen storage disease type Ib is functionally distinct from the bacterial hexose phosphate transporter, which operates as an obligatory phosphate:phosphate or G6P:phosphate exchanger.     

P2-purinergic control of liver glycogenolysis.

Purinergic agonists cause a dose-dependent activation of glycogen phosphorylase in isolated rat hepatocytes. Half-maximally effective concentrations are 5 X 10(-7)M for ATP, 2 X 10(-6)M for ADP, and about 5 X 10(-5) M for AMP and adenosine. This potency series indicates the presence of P2-purinergic receptors. The mode of action of ATP appears to be identical with that of the Ca2+-dependent glycogenolytic hormones angiotensin, vasopressin and alpha 1-adrenergic agonists. (1) They all require Ca2+ for phosphorylase activation; (2) they do not increase cyclic AMP levels; (3) they are susceptible to heterologous desensitization by vasopressin and phenylephrine; (4) they lower cyclic AMP concentrations in hepatocytes stimulated by glucagon, most probably mediated by an enhanced phosphodiesterase activity.     

Enzyme activity during the metabolism of glycogen. II. Cytochemical study of glycogen synthetase in the sensory cells of the tuberous organ of Gnathonemus petersii (Mormyridae).

Glycogen synthetase (2.4.1.11) forms I (independent or active) and D (dependent or passive) as well as the enzymes active in the transformation of the pathways, protein kinase and phosphatase transferase, were studied in the sensory cells and glycogen rich epidermal cells of the weakly electric fish Gnathonemus petersii (Mormyridae). For light microscopy an indirect cytochemical method which differentiated between glycogen originally present and that produced during incubation in the presence of UDPG was used. This differentiation was obtained by iodine, PAS and alpha and beta amylases. Glycogen synthetase is present in the sensory cells in the I and D forms. The epidermal cells only contain the D form. Protein kinase (active I yields D) has only been found in the sensory cells but phosphatase transferase (active D yields I) has been found in both the epidermal cells and the sensory cells, but only within certain organs. Electron microscopy studies of glycogen synthetase I and D and protein kinase were restricted to the sensory cells only. As with the light microscope it was possible to differentiate between native glycogen and newly formed glycogen. This was done using ultrathin sections and staining with uranyl acetate, lead citrate or by the PATAg reaction. It was possible from these observations to locate precisely the positions of these enzymes. In fact, glycogen synthetase I and D are found both in the sensory cytoplasm and in the sensory cavity with the polysaccharide filaments. Protein kinase is also abundant in the sensory cytoplasm especially in the periphery of the cell near the microvillary border.     

Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO₄, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg²⁺ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO₄ improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO₄ or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO₄ protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO₄ protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.     

THE LOCALIZATION OF GLYCOGEN IN THE SPERMATOZOA OF VARIOUS INVERTEBRATE AND VERTEBRATE SPECIES

With the periodic acid-thiosemicarbazide-silver proteinate procedure for the detection of polysaccharides in thin sections, glycogen is localized in the cavities of centrioles and basal bodies, within the axoneme (and surrounding it), in mitochondria, and in the "packing" cytoplasm of the middle piece of spermatozoa of several invertebrate and vertebrate species. The cytochemical localization of glycogen is verified by extraction with α-amylase solution. These findings establish the existence of stored glycogen in sperm. The polysaccharide presumably serves as an endogenous source of energy in the absence of extracellular metabolites, under either aerobic or anaerobic conditions. Other hypotheses on the physiological significance of intracellular glycogen stores in sperm are discussed. Sperm that store glycogen contain some enzymes of glycogen metabolism. In the presence of glucose-1-phosphate, ATP, and Mg⁺⁺ ions, an amylophosphorylase catalyzes the in vivo synthesis of glycogen. The newly formed product resembles γ-particles, and is digestible with α-amylase.     

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.     

ATP enhances cyclic AMP accumulation by intact P388 mouse lymphoma cells

One of the characteristics of malignant cells is a poor response to hormones and a low level of cyclic AMP. Whilst this is true of intact P388 mouse lymphoma cells, high levels of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity can be measured in particulate preparations of these cells. When ATP is added to the incubation medium of intact lymphoma cells, the cyclic AMP level is enhanced. This effect of ATP is not mediated by adenosine, nor is it enhanced by NaF. The ATP content of the lymphoma cells is much lower than that of CH23 Chinese hamster fibroblast and PCM3 hybrid cells, whose cyclic AMP levels are not affected by the presence of ATP. This suggests that adenylate cyclase, in the lymphoma cells, is bathed in a pool which is deficient in substrate. The substrate concentration of this pool is thought to be elevated by addition of ATP to the incubation medium with ATP, itself, crossing the plasma membrane.     

Activation of glycogenolysis in perfused rat livers by glucagon and metabolic inhibitors

The activation of hepatic glycogenolysis by glucagon and metabolic inhibitors was studied in isolated perfused livers from fed rats. Glucose production rates and phosphorylase activity were increased by all these agents. If iodoacetate (1 mM) and cyanide (1 mM) were infused simultaneously, glycogenolysis was activated to the same extent as by glucagon (1 nM). The effects of the hormone were additive to those of cyanide, but not to those of iodoacetate. When glycogen breakdown was maximally activated by cyanide plus glucagon, additional iodoacetate was inhibitory. The glucagon-induced release of cyclic AMP into the perfusate was partially suppressed by iodoacetate. The inhibitors caused various degrees of depletion of the tissue ATP content and parallel augmentation of the AMP levels. ADP rose to a lesser extent. Indirect evidence suggested that of a progressive lowering of the cellular ATP levels was accompanied by an inhibition of enzyme dephosphorylation as well as of phosphorylation processes. However, dephosphorylation appeared to be more sensitive to changes of the energy balance, resulting in an activation of phosphorylase in response to the metabolic inhibitors.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Cyclic adenosine 3',5'-monophosphate phosphodiesterase from Mucor rouxii: regulation of enzyme activity by phosphorylation and dephosphorylation.

Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg²⁺ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ ³²P]ATP resulted in a significant amount of [³²P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg²⁺ and was accompanied by the release of [³²P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.     

Liver metabolism in cold hypoxia: a comparison of energy metabolism and glycolysis in cold-sensitive and cold-resistant mammals

The effects of cold hypoxia were examined during a time-course at 2 °C on levels of glycolytic metabolites: glycogen, glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, lactate and energetics (ATP, ADP, AMP) of livers from rats and columbian ground squirrels. Responses of adenylate pools reflected the energy imbalance created during cold hypoxia in both rat and ground squirrel liver within minutes of organ isolation. In rat, ATP levels and energy charge values for freshly isolated livers were 2.54 mol·g^-1 and 0.70, respectively. Within 5 min of cold hypoxia, ATP levels had dropped well below control values and by 8 h storage, ATP, AMP, and energy charge values were 0.21 mol·g^-1, 2.01 mol·g^-1, and 0.17, respectively. In columbian ground squirrels the patterns of rapid ATP depletion and AMP accumulation were similar to those found in rat. In rat liver, enzymatic regulatory control of glycolysis appeared to be extremely sensitive to the decline in cellular energy levels. After 8 h cold hypoxia levels of fructose-6-phosphate decreased and fructose-1,6-bisphosphate increased, thus reflecting an activation of glycolysis at the regulatory step catalysed by phospho-fructokinase fructose-1,6-bisphosphatase. Despite an initial increase in flux through glycolysis over the first 2 min (lactate levels increased 3.7 mol·g^-1), further flux through the pathway was not permitted even though glycolysis was activated at the phosphofructokinase/fructose-1,6-bisphosphatase locus at 8 h, since supplies of phosphorylated substrate glucose-1-phosphate or glucose-6-phosphate remained low throughout the duration of the 24-h period. Conversely, livers of Columbian ground squirrels exhibited no activation or inactivation of two key glycolytic regulatory loci, phosphofructokinase/fructose-1,6-bisphosphatase and pyruvate kinase/phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Although previous studies have shown similar allosteric sensitivities to adenylates to rat liver phospho-fructokinase, there was no evidence of an activation of the pathway as a result of decreasing high energy adenylate, ATP or increasing AMP levels. The lack of any apparent regulatory control of glycosis during cold hypoxia may be related to hibernator-specific metabolic adaptations that are key to the survival of hypothermia during natural bouts of hibernation.Abbreviations DHAP dihydroxyacetonephosphate - EC energy charge - F1,6P₂ fructose-1,6-bisphosphate - F2,6P₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphatase - G1P glucose-1-phosphate - G6P glucose-6-phosphate - GAP glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - L/R lactobionate/raffinose-based solution - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PEPCK & PC phosphoenolpyruvate carboxykinase and pyruvate carboxylase - PFK phosphofructokinase; PK, pyruvate kinase - Q ₁₀ the effect of a 10 °C drop in temperature on reaction rates (generally, Q ₁₀=2–3) - TA total adenylates - UW solution University of Wisconsin solution (L/R-based)     

Cytophotometric analysis of glycogen, protein and DNA of a glycogen-storing rat hepatoma (N13) cell line.

This study examines the behavior of glycogen-storing rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G2/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N6,O2'-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G0/G1 and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Influence of some mononucleotides and their corresponding nucleosides on the metabolism of carbohydrates in the isolated rat diaphragm muscle.

1. The influence of ATP on glucose metabolism was studied in the isolated rat diaphragm; it was shown that ATP increases the oxidation of glucose and the aerobic conversion of glucose into lactate, whereas it decreases glycogen synthesis. There was no influence of ATP on the anaerobic formation of lactate from glucose. 2. A maximum effect of ATP on the oxidation of glucose (about 160% increase) was obtained in the presence of 10mm-ATP; in the presence of 2mm-ATP the effect was about 65%, and was approximately constant from 10 to 90min. incubation period. 3. In a phosphate-free tris-buffered medium the oxidation of glucose was considerably decreased, but the percentage stimulation by ATP was about the same as in a phosphate-buffered medium. 4. ATP was shown to increase the oxidation of fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 1,6-diphosphate and, to a much smaller extent, pyruvate. 5. ADP stimulated the oxidation of glucose to the same extent as ATP at a concentration of 2mm and the effect with AMP was only slightly less; IMP and adenosine had only a small stimulatory effect at this concentration, whereas inosine had no effect.     

Cytophotometric analysis of glycogen,protein and DNA of a glycogen-storing rat hepatoma (N13) cell line

This study examines the behavior of glycogenstoring rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G₂/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N⁶,O²-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G₀/G₁ and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Cytochemistry and distribution of polysaccharides in an electroreceptor: the tuberous organ of Gnathonemus petersii (Mormyrids).

The polysaccharides were studied in an electroreceptor organ, the tuberous organ of Gnathonemus petersii (Mormyridae). Histochemical methods (P.A.S., alcian blue, toluidine blue and iron colloidal reactions) allowed us to demonstrate the existence of glycogen in the sensory cytoplasm, and P.A.S. positive polysaccharides in the sensory cavity. The polysaccharides were shown to be amylase proof; they display an acidity due to the existence of sulphated radicals. The histochemical study was completed by a cytochemical analysis: a treatment with thiocarbohydrazide (TCH) according to the Thiery's method. This method allowed us to estimate the glycogen concentration, its localization, and relationship with cellular organites within the sensory cytoplasm, as well as to differentiate the highly glycogenous type II cells of the platform from the other accessory cells (Derbin and Szabo, 1968). After a treatment for 20 hours with TCH, silver stained grains were visible on the polysaccharide filaments of the sensory chamber, between the microvilli and the vacuoles of the epidermal cells lining to the sensory cavity. Silver grains coated the outer surface of the microvilli. Such polysaccharides were not identical to the filamentous polysaccharides of the cavity. In order to determine the cytochemical localization of the polysaccharide acid groups, sections were stained with iron salts. The colloidal iron constitutes a deposit opaque to electrons and located both on the filamentous polysaccharides of the sensory cavity and in the vacuoles of the epidermal cells, indicating that only these filamentous polysaccharides display acid radicals.