A UDP-glucose derivative is required for vacuolar autophagic cell death

Autophagic cell death in Dictyostelium can be dissociated into a starvation-induced sensitization stage and a death induction stage. A UDP-glucose pyrophosphorylase (ugpB) mutant and a glycogen synthase (glcS) mutant shared the same abnormal phenotype. In vitro, upon starvation alone mutant cells showed altered contorted morphology, indicating that the mutations affected the pre-death sensitization stage. Upon induction of cell death, most of these mutant cells underwent death without vacuolization, distinct from either autophagic or necrotic cell death. Autophagy itself was not grossly altered as shown by conventional and electron microscopy. Exogenous glycogen or maltose could complement both ugpB^- and glcS^- mutations, leading back to autophagic cell death. The glcS- mutation could also be complemented by 2-deoxyglucose that cannot undergo glycolysis. In agreement with the in vitro data, upon development glcS^- stalk cells died but most were not vacuolated. We conclude that a UDP-glucose derivative (such as glycogen or maltose) plays an essential energy-independent role in autophagic cell death.     

Regulation of renal glycogen synthase interconversion of two forms in vitro

Glycogen synthase (UDP glucose: glycogen α-4-glucosyltransferase, EC 2.4. 1.11) from rat kidney was stimulated 4- to 5-fold by glucose 6-phosphate. The for glucose 6-phosphate stimulation was about 0.45 mM. Glycogen synthase was not evenly distributed throughout the kidney. Total synthase activity was greatest in the outer cortex and cortico-medullary junction and least in the inner medulla. Glucose 6-phosphate stimulation was greatest in the outer cortex and least in the inner medulla. Glycogen synthase in crude homogenates was not complexed with glycogen and eluted from Sepharose 6-B with an apparent molecular weight of about 390 000.Renal glycogen synthase appeared to exist in two interconvertible forms, synthase I (activity in the absence of glucose 6-phosphate) and synthase D (requires glucose 6-phosphate for activity). The conversion of synthase D to I (synthase D phosphatase) was inhibited by F⁻, glycogen, ATP, Mn²⁺, and Co²⁺. The conversion was not altered by mercaptoethanol, AMP, Mg²⁺, or Ca²⁺. The conversion of synthase I to D (synthase I kinase) required ATP-Mg and was stimulated by cyclic AMP.It was suggested that the interconversion of renal glycogen synthase involved a phosphorylation-dephosphorylation. The significance of glycogen synthase interconversion to the regulation of renal glycogen synthesis is discussed.     

On the process of activation and inactivation of dog skeletal muscle glycogen synthase.

The presence of additional forms of glycogen synthase (UDPG: alpha-1,4-glucan alpha-4-glucosyltransferase) besides the I form (independent on glucose-6-P for activity) and the D form (dependent on glucose-6-P for activity) long ago described, is inferred from patterns of their interconversions obtained by processes of phosphorylation and dephosphorylation. An intermediate form more phosphorylated than the I form and less than the D form, which is completely inactive in these assay conditions, and a superphosphorylated form, more phosphorylated than the D form and also inactive even in presence of 0.01 M glucose-6-P are described.     

Role of nuclear glycogen synthase and cytoplasmic UDP glucose pyrophosphorylase in the biosynthesis of nuclear glycogen in HD33 Ehrlich-Lettre ascites tumor cells

Biochemical and autoradiographic evidence show both glycogen synthesis and the presence of glycogen synthase (UDP glucose [UDPG]: glycogen 4-alpha-D-glucosyltransferase; EC 2.4.1.11) in isolated nuclei of Ehrlich-Lettré mouse ascites tumor cells of the mutant subline HD33. 5 d after tumor transplantation, glycogen (average 5-7 pg/cell) is stored mainly in the cell nuclei. The activity of glycogen synthase in isolated nuclei is 14.5 mU/mg protein. At least half of the total cellular glycogen synthase activity is present in the nuclei. The nuclear glycogen synthase activity exists almost exclusively in its b form. The Km value for (a + b) glycogen synthase is 1 x 10(-3) M UDPG, the activation constant is 5 x 10(-3) M glucose-6-phosphate (Glc-6-P). Light and electron microscopic autoradiographs of isolated nuclei incubated with UDP-[1-3H]glucose show the highest activity of glycogen synthesis not only in the periphery of glycogen deposits but also in interchromatin regions unrelated to detectable glycogen particles. Together with earlier findings on nuclear glycogen synthesis in intact HD33 ascites tumor cells (Zimmermann, H.-P., V. Granzow, and C. Granzow. 1976. J. Ultrastruct. Res. 54:115-123), the results of tests on isolated nuclei suggest a predominantly appositional mode of nuclear glycogen deposition, without participation of the nuclear membrane system. In intact cells, synthesis of UDPG for nuclear glycogen synthesis depends on the activity of the exclusively cytoplasmic UDPG pyrophosphorylase (UTP: alpha-D-glucose-1-phosphate uridylyltransferase; EC 2.7.7.9). However, we conclude that glycogen synthesis is not exclusively a cytoplasmic function and that the mammalian cell nucleus is capable of synthesizing glycogen.     

Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds.

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.     

Cross‐species functional complementation of cellulose synthase during the development of cellular slime molds

Cellulose is a major and important component of the extracellular matrix during the development of Dictyostelium discoideum. Upon starvation, solitary amoebae of D. discoideum gather and form fruiting bodies in which cells differentiate into stalk cells and spores. The stalk tubes and walls of spores and stalk cells are made of cellulose. In the genus Acytostelium, however, all cells are destined to become spores and the stalks comprise only a cellulose tube, suggesting species‐specific regulation of cellulose synthesis. In this study, we cloned a putative cellulose synthase gene (cesA) of Acytostelium subglobosum and performed comparative analyses with the D. discoideum cellulose synthase gene (dcsA). Although the deduced amino acid sequences were highly conserved between cesA and dcsA, the numbers of transmembrane spans preceding the catalytic domain were dissimilar; 2 and 3, respectively. Since ectopic expression of cesA in dcsA^?null cells failed to restore the developmental defects of the mutant, we constructed a series of chimerical genes for complementation analyses and found that the catalytic domain of cesA was functional in D. discoideum cells if the preceding transmembrane region was swapped with dcsA. The non‐functional products that contained the cesA‐derived transmembrane region were localized to lysosomes. These results indicate that the transmembrane region of cellulose synthase is essential for proper accumulation of cellulose during the development of D. discoideum and that its differential localization in A. subglobosum may be related to the characteristic morphogenesis in this species.     

Liver glycogen metabolism in endotoxin shock. I. Endotoxin administration decreases glycogen synthase activities in dog livers

The effects of E. coli endotoxin administration on hepatic glycogen content and glycogen synthase activities in dogs were studied. Liver glycogen content was decreased by 80% 2 hr after endotoxin injection. When enzyme preparations were preincubated at 25 degrees C for 3 hr prior to their assays, 75% of total glycogen synthase was in I form in control dogs. Under such conditions, endotoxin administration decreased the percentage I activity from 75 to 37%; decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 62.2 and 35.3%, respectively; decreased the Vmax and Km for UDP-glucose for glycogen synthase I by 75.6 and 15.6%, respectively; increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 126% at high (10 mM) and by 18-fold at low (1 mM) UDP-glucose concentration; increased the percentage D activity from 24 to 72%; decreased the I50 for ATP for the inhibition of total glycogen synthase by 49.7%; decreased the I50 for ATP for the inhibition of glycogen synthase I by 26.4%; and decreased the percentage I activity from 78 to 33% at ATP concentrations below 6 mM. When enzyme preparations were not preincubated prior to their assays, 90% of total glycogen synthase was in D form in control dogs. Under such conditions, endotoxin administration decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 47.1 and 33.3%, respectively, and increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 24.2% at high (10 mM) and by 106% at low (1 mM) UDP-glucose concentration. From these results, it is clear that endotoxin administration greatly impaired hepatic glycogenesis by decreasing the activity of glycogen synthase; this impairment is at least in part responsible for the depletion of liver glycogen content in endotoxin shock. Kinetic analyses revealed that the decrease in the activity of glycogen synthase in endotoxic shock is a result of a decrease in the interconversion of this enzyme from inactive to active form and an increase in the interconversion from active to inactive form.     

Purification and steady state kinetic mechanism of glycogen synthase-D from human polymorpho-nuclear leukocytes

Summary The authors' work on the purification and steady state kinetic investigation of the enzyme glycogen synthase D (UDP-glucose: glycogen 4--glucosyl-transferase, EC 2.4.1.11) from human polymorphonuclear leukocytes is reviewed. The main features of the kinetic mechanism for catalysis of the reaction UDPG + glycogen_n UDP + glycogen_(n+1) are: (i) Lineweaver-Burk plots in both substrates are linear, exhibiting intersecting patterns; (ii) UDP is a competitive, respectively noncompetitive, inhibitor towards the substrates UDPG and glycogen; (iii) the essential activator glucose-6-phosphate (G-6-P) showed an intersecting pattern towards glycogen and an equilibrium ordered pattern towards UDPG. These features identify in this case the mechanism as a rapid equilibrium random bi-bi mechanism, with G-6-P adding to the enzyme prior to the substrate UDPG. New results on the influence of the modifiers NaCl, Ca⁺⁺, Mn⁺⁺, Mg⁺⁺, HPO₄ ^–-, SO₄ ^–-, and ATP on the enzyme are reported. Interpreting the observations in terms of the established mechanism, the following results are obtained: The effect of salt (NaCl) is nonspecific and fairly small, probably reflecting a general action of the electrolyte medium on the conformation of the enzyme. Divalent cations affect only the rate limiting step, i.e. the interconversion of the quaternary enzyme-substrate-activator complexes. The anions interact exclusively with the G-6-P binding site of the enzyme. The dissociation constants for the enzyme-modifier complexes are determined, and a kinetic mechanism for the action of the anions is proposed, leading to activation or inhibition, depending on the concentration of G-6-P.An invited article     

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: Complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene

Summary Three cellulose-negative (Cel^-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize (1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.     

Rat adipose tissue glycogen synthase. Evidence for multiple discrete kinetic species and their interconversion.

Rat adipose tissue glycogen synthase has been kinetically characterized. The classical D form has an apparent Km for UDP-glucose of 0.7 mM and 0.4 mM in the absence and presence of glucose 6-phosphate, respectively. The apparent Ka for glucose 6-phosphate is 0.6 mM. The effect of glucose 6-phosphate on the D form is to enhance the Vmax 7-fold. The I form is also affected by glucose 6-phosphate (Ka, 0.025 mM) but the Vmax is increased only by 20%; apparent Km values for UDP-glucose are 0.4 mM and 0.045 mM in the absence and presence of glucose 6-phosphate, respectively. In addition, two new kinetically distinguishable forms have been observed. The first, designated glycogen synthase Q, arises from an Mg2+ATP-dependent deactivation of the I form. The apparent Km values of glycogen synthase Q for UDP-glucose are identical with those of the I form; however, the apparent Ka for glucose 6-phosphate (0.2 mM) is 8-fold higher than that for the I form and one-third that for the D form. Preparations from fasted or diabetic rats contain a form of glycogen synthase, designated glycogen synthase X, that has a much lower affinity for glucose 6-phosphate than the D form (apparent Ka, 3 mM); the apparent Km values for UDP-glucose are similar to those of the D form (0.7 mM and 0.3 mM in the absence and presence of glucose 6-phosphate, respectively). In preparations from fasted rats a stepwise Mg2+-dependent conversion was demonstrated of synthase X to D to Q to I; this sequential conversion was reversed on incubation with Mg2+ATP. In preparations from fed rats, synthase Q could be generated either by limited activation (from the D form) or, after conversion to the I form, by deactivation with Mg2+ATP. However, even prolonged incubation with Mg2+ATP failed to generate the D (or X) form.     

Purification and properties of the glucose-6-phosphate-dependent form of human placental glycogen synthase.

Glycogen synthase in the glucose-6-phosphate (glucose-6-P)-dependent form was purified over 10,000-fold from an extract of term human placenta. The purified enzyme shows a single protein band on polyacry1amide-gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme activity in the presence of glucose-6-P is increased by the single addition of Mg²⁺, Ca²⁺, or Mn²⁺ and is reduced by the addition of either sulfate or phosphate. Addition of either Mg²⁺, Ca²⁺, or Mn²⁺ relieves the inhibition by sulfate or phosphate. The enzyme activity in the absence of glucose 6-P is greatly increased by the addition of MnSO₄, CoSO₄, and NiSO₄ and is increased to a lesser extent by MgSO₄, CaSO₄, and FeSO₄. The activation of the glucose-6-P-dependent form of the enzyme by these metal sulfates in the absence of glucose-6-P has never been reported. MnSO₄, which shows homotropic cooperativity, is the best activator among the various metal sulfates tested. The human placental glucose-6-P-dependent form of glycogen synthase (D form) can be converted to the glucose-6-P-independent form (I form) of the enzyme by incubating the partially purified glycogen synthase, which is copurified with synthase phosphatase, with Mn²⁺. This conversion can be reversed by the addition of cyclic AMP-dependent protein kinase. The synthase D to synthase I converting system from human placenta is unique in its stringent requirement for Mn²⁺.     

Duplicated gene clusters suggest an interplay of glycogen and trehalose metabolism during sequential stages of aerial mycelium development in Streptomyces coelicolor A3(2)

DNA sequencing and operon disruption experiments indicate that the genes glgBI and glgBII, which code for the two developmentally specific glycogen branching enzymes of Streptomyces coelicolor A3(2) each form part of larger duplicated operons consisting of at least four genes in the order pep1-treS-pep2-glgB. The sequences of the TreS proteins are 73% identical (93% similar) to that of an enzyme that converts maltose into trehalose in Pimelobacter, a distantly related actinomycete; and the Pep1 proteins show relatedness to the α-amylase superfamily. Disruptions of each operon have spatially specific effects on the nature of glycogen deposits, as assessed by electron microscopy. Upstream of the glgBI operon, and diverging from it, is a gene (glgP) that encodes a protein resembling glycogen phosphorylase from Thermatoga maritima and a homologue in Mycobacterium tuberculosis. These three proteins form a distinctive subgroup compared with glycogen phosphorylases from most other bacteria, which more closely resemble the enzymes from eukaryotes. Diverging from the glgBII operon, and separated from the pep1 gene by two very small ORFs, is a gene (glgX) encoding a probable glycogen debranching enzyme. It is suggested that most of these gene products participate in the developmentally modulated interconversion of immobile, inert glycogen reservoirs, and diffusible forms of carbon, both metabolically active (e.g. glucose-1-phosphate generated by glycogen phosphorylase) and metabolically inert but physiologically significant (trehalose). Received: 12 November 1999 / Accepted: 31 January 2000     

The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.

The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Glycogen synthase in diabetic rat skeletal muscle: activation by insulin

Summary Glycogen synthase in skeletal muscle of 3-day alloxan-diabetic rats was found to be in a less active state than in normal muscle. Both the activity ratio (activity without G6P divided by activity with 7.2 mM G6P at 4.4 mM UDPG, pH 7.8) and fractional velocity (activity with 0.25 mM G6P divided by activity with 10 mM G6P at 0.03 mM UDPG, pH 6.9) were significantly lower in the diabetic tissue. Correspondingly, the S_0.5 for UDPG and A_0.5 for G6P were significantly higher in diabetic tissue, suggesting decreased affinity for substrate and activator, respectively. The kinetic changes in the diabetic synthase were identical whether the alloxan-treated animals were maintained on insulin for 7 days prior to withdrawal for 3 days, or studied 3 days immediately after alloxan treatment. The diabetes-induced changes in synthase could be reversed by injecting the diabetic rat with insulin 10 min prior to sacrifice. After insulin treatment, the S_0.5 for UDPG and A_0.5 for G6P decreased to control levels or lower and the activity ratios and fractional velocities increased to control levels or higher.The activity of glycogen synthase phosphatase was not decreased in diabetic skeletal muscle. This observation, coupled with the rapid response of the diabetic synthase to in vivo insulin treatment, suggests that, unlike the phosphatase in cardiac muscle and liver, the glycogen synthase phosphatase in skeletal muscle is not altered by the diabetic state.Abbreviations UDPG uridine diphosphoglucose - G6P glucose 6-phosphate - EDTA ethylene diamine tetraacetic acid - IP intraperitoneally - MOPS morpholinopropane sulfonic acid - -ME -mercaptoethanol - V_G6P calculated velocity of the enzyme in the presence of infinite G6P concentration - V_UDPG calculated velocity of the enzyme in the presence of infinite UDPG concentration     

Intracellular and intramitochondrial binding of lanthanum in dark degenerating midgut cells of a collembolan (Insect)

Summary 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 and 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 ID) has only been found in the sensory cells but phosphatase transferase (active DI) 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.