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1.
HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.  相似文献   

2.
When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.  相似文献   

3.
Glycogen synthase from bovine adipose tissue has been kinetically characterized. Glucose 6-phosphate increased enzyme activity 50-fold with an activation constant (A0.5) of 2.6 mm. Mg2+ reversibly decreased this A0.5 to 0.75 mm without changing the amount of stimulation by glucose 6-phosphate. Mg2+ did not alter the apparent Km for UDP-glucose (0.13 mm). The pH optimum was broad and centered at pH 7.6. The glucose 6-phosphate activation of the enzyme was reversible and competitively inhibited by ATP (Ki = 0.6 mm) and Pi(Ki = 2.0 mm). The use of exogenous sources of glycogen synthase and glycogen synthase phosphatase suggests that (i) adipose tissue glycogen synthase phosphatase activity in fed mature steers is low or undetectable, and (ii) endogenous bovine adipose tissue glycogen synthase can be activated to other glucose 6-phosphate-dependent forms by addition of adipose tissue extracts from fasted steers or fed rats.  相似文献   

4.
A glucosidase preparation with activity toward certain glucose-containing oligosaccharides was partially purified from calf liver membranes by Triton X-100 solubilization and DEAE-cellulose and hydroxylapatite chromatography. The enzyme preparation hydrolyzed the glucose residues from (glucose)1,(mannose)9(N-acetylglucosamine)1, and (glucose)2(mannose) 9(N-acetylglucosamine)1 but was totally inactive toward (glucose)3(mannose)9(N-acetylglucosamine) 1. In contrast, crude membrane preparations of the calf liver were active toward all three substrates. The partially purified enzyme had a pH optimum of 6.7 and was very unstable in the absence of added 20% glycerol. The rate of glucose release from the one-and two-glucose-containing oligosaccharides was significantly decreased when four or five of the mannose residues were first removed from the substrate. The release of glucose from (glucose)1(mannose)9(N-acetylglucosamine)1 was inhibited by p-nitrophenyl-α-d-glucoside much more effectively than by p-nitrophenyl-β-d-glucoside, suggesting that this glucose residue may be linked α to the mannose residue. We conclude that during oligosaccharide processing at least two different glucosidases are involved in glucose removal.  相似文献   

5.
—Major components of the energy reserves of the isolated superior cervical ganglion (ATP, phosphocreatine, glucose, glycogen and lactate) were measured under aerobic and anaerobic conditions. Complete anaerobiosis was maintained by incubation in mineral oil through which N2 had been bubbled. From the initial rate of change in the energy reserves, a metabolic rate was calculated which would be equivalent to the consumption of 93 m-moles of O2 per kg per hour. Under aerobic conditions (oxygenated moist chamber) a similar metabolic rate was calculated. In contrast to the anaerobic state, initial energy expenditure was almost exclusively at the expense of glucose. Continuous supramaximal stimulation in O2 increased energy expenditure by a factor of three; both glucose and glycogen were utilized from the outset, and lactate accumulated in the initial periods. Ganglionic transmission failed in both resting and stimulated states in spite of the continued presence of very substantial levels of ATP and phosphocreatine. Failure seemed to be associated not with ATP depletion but rather with the complete disappearance of glucose and glycogen.  相似文献   

6.
Using cytochemical and Förster resonance energy transfer (FRET) methods, the structure of glycogen was studied in rat hepatocytes during starvation and in some time intervals after the peroral administration of glucose to the animals. Hepatocytes were stained with a fluorescent variant of PAS reaction on object glasses. The staining of preparations for 40 min with ethidium bromide-SO2 (EtBr-SO2) revealed the labile fraction (LF) of glycogen, while their subsequent staining with auramine-SO2 (Au-SO2) for 50 min revealed the stable fraction (SF) of glycogen in cells. The total glycogen content (LF + SF) in hepatocytes at various stages of rat refeeding was determined using a cytofluorimeter; then, in the same cells, the FRET efficiency was measured. Recording FRET at several sites of cells was performed using a Leica TCS SP5 laser scanning confocal microscope by using the FRET Acceptor Photobleaching (FRET AB) procedure. In this procedure, auramine was used as the donor (D), while ethidium bromide was used as the acceptor (A). The efficiency of FRET in the course of rat refeeding with glucose has been shown to change from 10 to 14%, and the glycogen structure markedly affects the value of this parameter. It is found that, in cells of starved rats and in early terms after the administration of glucose, the FRET efficiency correlates with the A/D ratio, which reflects the degree of filling of external tiers of glycogen molecules with glucose residues. At later terms of refeeding, this correlation is either less pronounced or completely absent. It has been established that, at the same A/D value, the FRET efficiency can change by three to four times. Since the probability of energy transduction from D to A is proportional to 1/R6, where R is the distance between D and A. These fluctuations of the FRET efficiency mean that the glycogen molecules have the labile structure, in which chains of glycoside residues can deviate from its axis at a distance of about a half of their diameter.  相似文献   

7.
The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with Pi or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to Pi changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to Pi. Arsenate is a competitive inhibitor with respect to Pi. The Km for AMP increases in the presence of UDPG, and decreases with increasing concentrations of Pi or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for Pi, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.  相似文献   

8.
Right-handed RNA duplexes of (CG)n sequence undergo salt-induced helicity reversal, forming left-handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)2 determined in 6 M NaClO4. This is the first nucleic acid motif determined at such high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6(n)-H5′/H5″(n)-H8(n+1)-H1′(n+1)-H6(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4–7°), whereas GpC steps have large negative values (−61°). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2′-OH groups, and the major groove are well defined. The 2′-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2′-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.  相似文献   

9.
Summary Fibrin-enrobed, commercially produced glycogen was treated, without prior glutaraldehyde fixation, to a form of post-fixation with solutions of OsVIIIO4 or with a mixture of either OsVIIIO4 plus K3FeIII(CN)6 or K2OsVIO4 plus K4FeII(CN)6.Only the last mixture gave constrast staining of the glycogen in unstained ultrathin sections. The first mixture rendered the glycogen just barely visible but the glycogen contrast was increased by lead staining. The glycogen treated with the OsVIIIO4 solution was not contrast stained and was just observable after lead staining.Qualitative X-ray microanalysis of the glycogen in the ultrathin sections confirmed the presence of osmium and iron in the glycogen treated with both mixtures. The glycogen treated with OsVIIIO4 alone was difficult to analyse.Quantitative X-ray microanalysis showed that, in the glycogen treated with the OsVIIIO4 mixture plus K3FeIII(CN)6, the mean atomic osmium to iron ratio was 15. In the glycogen treated with K2OsVIO4 plus K4FeII(CN)6 this ratio was 117. However, the mean net osmium intensity in the latter case was 15 times higher than in the former case and for the iron even 40 times higher.The Unit for Analytical Electron Microscopy was established by collaboration between the Erasmus University of Rotterdam (W. C. de Bruijn), the University of Leiden and the Organization for Health Research TNO. The analytical microscope was purchased with funds from the Netherlands Organization for Pure Scientific Research (ZWO).  相似文献   

10.
A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen + phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml−1. The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.  相似文献   

11.
12.
《Inorganica chimica acta》1987,129(2):205-216
The synthesis, spectroscopic and magnetic properties of two nickel clusters are described, Ni8(NCS)8(Hahmt)6(H2ahmt)4(ahmt)(H2O)12 (A) and Ni4(Hahmt)4(H2ahmt)2(NCS)4(H2O)4 (B) (H2ahmt = 4-amino-3,5-bis(hydroxymethyl)-1,2,4-triazole). The X-ray structure of A has been determined. The compound crystallizes in the space group C2/c, a = 25.458(2), b = 15.466(2), c = 26.959(3) Å, β = 90.648(5)°. The structure was refined to R = 0.108 for 4169 observed reflections. The structure consists of two Ni4O4 cubane-type clusters, each consisting of four nickel atoms, three singly deprotonated and one doubly deprotonated ligands. Hahmt coordinates as a bidentate chelating ligand through its triazole-N1 and its 3-oxymethyl-part. The doubly deprotonated ligand chelates in a bis-bidentate manner to two Ni4 clusters. In this way dumbbell-like pairs of ligand bridged cubanes are formed. Along two opposite diagonals of the Ni4O4 cubanes a neutral ligands, coordinating through its triazole-N1,N2 atoms, forms a bridge between two nickel ions. The NiN3O3 chromophore is completed by a monodentate N-coordinating thiocyanate anion. Compound B, for which only partial structure determination was possible, has the same Ni4O4 cubane-type cluster, however, without the bridging ligand between the cubanes. The two types of NiNi bridges result in two unequivalent superexchange pathways. In the compounds both ferromagnetic and antiferromagnetic interaction pathways are present, resulting in an overall antiferromagnetic behaviour. The nature of the interaction for the different pathways is related to the observed NiONi angles. The intercluster exchange is much larger through the triazole bridge in A than it is in B, where only hydrogen bridges keep the clusters together.  相似文献   

13.
Extraction of defatted garlic bulbs with hot water yielded a mixture of polysaccharides containing pectic acid, a D-galactan, and a fructan component. The pectic acid was partially removed as calcium pectate, and the galactan-enriched portion was separated by fractional precipitation with alcohol; on concentration and several fractionations, the supernatant liquor furnished the fructan component, which contained fructose (94.4%) and glucose (4.3 %). Methanolysis and hydrolysis of the permethylated fructan gave (a) 1,3,4,6-tetra-O-methyl-D-fructose, (b) 2,3,4,6-tetra-O-methyl-D-glucose, (c) 2,4,6-tri-O-methyl-D-glucose, and (d) 3.4,6-tri-O-methyl-D-fructose in the ratio (a + b): (d) = 1:20.3. On periodate oxidation, the fructan reduced one molar equivalent of the oxidant per hexosyl residue, and liberated one molar equivalent of formic acid per 51 hexosyl residues. On Smith degradation, the major product was glycerol, and ~2 % of the glucose survived. From these results, and from the fact that the fructan is hydrolyzed by β-D-fructofuranosidase, a linear, inulin-type of structure is suggested for it.  相似文献   

14.
The structure of glycogen phosphorylase alpha at 2.5 A resolution   总被引:10,自引:0,他引:10  
The structure of the glucose-inhibited form of glycogen phosphorylase a has been determined at a resolution of 2.5 Å. With the aid of the primary sequence derived by Titani et al. (1977) for this enzyme, we have constructed an atomic model of the 97,400 molecular weight monomer. A substantial improvement in the electron density map over that reported previously (Fletterick et al., 1976b) was achieved by extension of the data set to 2.5 Å and the inclusion of three additional “heavy-atom” derivatives in the phasing procedure. Main-chain and side-chain electron density are clearly resolved in the map, allowing an unambiguous correlation with the published primary structure. The course of the polypeptide backbone in the C-terminal half of the molecule has been modified at two positions from that reported in the 3.0 Å resolution interpretation.The enzyme is clearly organized into two domains, both with αβ packing topology. The catalytic site lies in a crevice at the interface between the two domains. α-d-Glucose, which stabilizes the inactive (T) conformation in the parent crystal, is bound at this site in the C(6′) chair equatorial conformation within 6 Å of the pyridoxal phosphate coenzyme which is covalently bound through the ?-amino group of lysine 679.The larger, N-terminal domain is differentiated by folding architecture and tertiary contacts into two lobes or subdomains which share the same β-sheet backbone: the predominantly helical glycogen storage (maltoheptaose binding) lobe and the N-terminal subdomain. The latter is involved in a variety of protein-protein interactions with the monomer related by the 2-fold axis of the physiological dimer, and contains the serine 14-phosphate moiety and the AMP (positive effector) binding site. The core of the second domain is the complex (βαβ)′ folding unit previously characterized as the nucleotide binding fold (Rao &; Rossmann, 1973).  相似文献   

15.
The crystal structure of potassium hydroxide complexed amylose, obtained by heterogeneous deacetylation of amylose triacetate, has been determined through a combined stereochemical structure-refinement and X-ray diffraction-analysis. The structure crystallizes in an orthorhombic unit-cell with parameters a  8.84, b  12.31, and c (fiber repeat)  22.41 Å, and with P212121 symmetry. The conformation of the amylose chain is a distorted, left-handed helix with 6 d-glucose residues per turn. Each three-residue asymmetric unit is complexed with one molecule of potassium hydroxide and three molecules of water. The K+ ion coordinates with four oxygen atoms of the amylose chain and with two other oxygen atoms, and this coordination is probably the cause for the more-extended amylose chain-conformation than would be predicted from a φ, ψ map. The distortions in the chain are primarily manifested by different O-6 rotations and by slightly different bridge and φ, ψ angles for the individual residues. The structure is extensively hydrogen bonded, although largely through water molecules, which accounts for its ready water solubility. The left-handed conformation of the chain in this structure is consistent with the conformations of amylose triacetate and V-amylose, both of which are left-handed.  相似文献   

16.
Previous reports have demonstrated the incorporation of glucose from ADP-glucose into methanol-insoluble and TCA-insoluble fractions in cell extracts of Escherichia coli in the absence of added primer α-glucan. This activity is reduced 6- to 76-fold in cell extracts of three independently isolated glycogen synthase-deficient mutants of E. coli B. Homogeneous preparations of E. coli B glycogen synthase catalyze incorporation of glucose into both methanol- and TCA-insoluble fractions in the absence of added primer. Since glycogen synthase catalyzes these reactions, it is not necessary to propose a protein acceptor glucose or a unique ADP-glucose-glycosyl transferase to catalyze formation of the glucoprotein in E. coli cell extracts to explain glucose incorporation into TCA-insoluble material (R. Barengo et al. (1975) FEBS Lett.53, 274–278). The incorporation of glucose into methanol-and TCA-insoluble fractions is stimulated by 0.25 m citrate and by branching enzyme. Citrate reduces the Km for the primer, glycogen, about 11- to 15-fold. Branching enzyme can also reduce the concentration of primer required for incorporation of glucose into methanol-insoluble material. The simultaneous presence of both 0.25 m citrate and branching enzyme enables the glycogen synthase reaction rate to proceed at 30% the maximal velocity at a primer concentration of 1 μg/ml. Incorporation of glucose into methanol- or TCA-insoluble material in the absence of primer is completely inhibited by adding α-amylase. Furthermore, incorporation into methanol- or TCA-insoluble material is reduced 13- to 16-fold relative to the reaction occurring in the presence of primer when glycogen synthase is pretreated with glucoamylase and α-amylase. Previous results show that homogeneous preparations of glycogen synthase contain glucan. Heat-denatured glucogen synthase can act as a primer for the glycogen phosphorylase and glycogen synthase reactions. Both the TCA- and methanol-insoluble products form I2-glucan complexes with wavelength maxima of about 580–590 nm and 610–615 nm, respectively, suggesting that they are mainly linear chain glucans. The products are completely solubilized with α-amylase. The TCA-insoluble product is not solubilized by pronase treatment. The above results strongly suggest that previous reports on formation of glucoprotein primer for glycogen synthesis or on de novo glycogen synthesis in various similar systems is due to endogenous glucan associated with glycogen synthase rather than formation of glucoprotein which then acts as primer for glycogen synthesis.  相似文献   

17.
Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1–GL) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1–GL complex, suppressing glycogen synthesis. Consequently, the interaction of GL with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the GL–phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the GL protein. The resulting GLY284F/Y284F mice display hepatic PP1–GL activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of GL by phosphorylase a operates in vivo. GLY284F/Y284F and GLY284F/+ mice display improved glucose tolerance compared with GL+/+ littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the GL–phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the GLY284F/Y284F mice lose more body weight and display decreased blood glucose levels in comparison with their GL+/+ littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of GL may prevent futile glucose–glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1–GL by phosphorylase a in mammals.  相似文献   

18.
Amylose was oxidized with 0.1–0.2 mol of periodate per glucose residue (G), and then reduced with sodium borohydride or borotritide to give an oxidized-reduced amylose of low degree of modification. Mild acid hydrolysis gave erythritol, 2-O-α-d-glucosyl-l-erythritol, higher homologs, and other products. Extensive action of porcine-pancreatic amylase on the polymer gave, besides d-glucose and maltose, oligosaccharides containing one or more oxidized-reduced (modified, M), acyclic residues. The enzymic products containing only one oxidized-reduced residue were identified as a modified tetrasaccharide (MG3) and a modified pentasaccharide (MG4). Structures of MG3 and MG4 were identified by a combination of enzymic and chemical approaches. With glucoamylase, MG4 was converted into MG plus d-glucose, whereas MG3 was totally resistant. On mild acidic hydrolysis, MG3 was converted into 2-O-α-d-glucosyl-d-erythritol plus maltose. These results indicate that MG3 is G-M-G-G and that MG4 is G-G-M-G-G. In principle, MG4 could occupy the five d-glucose residue, substrate-binding site of porcine-pancreatic amylase in such a way that M, the acyclic structure replacing a d-glucose residue, is placed just to the “left” of the catalytic site. The modified structure, being very vulnerable to acidic hydrolysis, might then be expected to be very readily attacked by the amylase, but in fact, it is not.  相似文献   

19.
The metabolism of rat retina was found to be sensitive to the concentration of the carbon dioxide-bicarbonate buffer system. Increasing the carbon dioxide from 1 per cent to 5 per cent at constant pH nearly doubled both respiration and glycolysis. Increasing the carbon dioxide at constant pH from 5 per cent to 20 per cent had no effect on glycolysis, but depressed the Q OO2 from 31 to 19. In a medium containing glucose and the 1 per cent carbon dioxide-bicarbonate buffer, the addition of succinate increased the Q OO2 from 12 to 26, without affecting glycolysis. In a medium containing glucose and phosphate, succinate had no significant effect.  相似文献   

20.
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.  相似文献   

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