scholarly journals Identification of Binding Sites on Protein Targeting to Glycogen for Enzymes of Glycogen Metabolism

2000 ◽  
Vol 275 (45) ◽  
pp. 35034-35039 ◽  
Author(s):  
Noel M. Fong ◽  
Timothy C. Jensen ◽  
Ami S. Shah ◽  
Nita N. Parekh ◽  
Alan R. Saltiel ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Targeting ◽  
Glycogen Metabolism

scholarly journals Glycogen Metabolism as a Marker of Astrocyte Differentiation

2009 ◽  
Vol 30 (1) ◽  
pp. 51-55 ◽  
Author(s):  
JF Brunet ◽  
I Allaman ◽  
PJ Magistretti ◽  
L Pellerin
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Bovine Serum ◽  
Leukemia Inhibitory Factor ◽  
Protein Targeting ◽  
Fetal Bovine Serum ◽  
Glycogen Metabolism ◽  
Ciliary Neurotrophic Factor ◽  
Astrocytic Differentiation ◽  
Astrocyte Differentiation ◽  
Fetal Bovine

Glycogen is a hallmark of mature astrocytes, but its emergence during astrocytic differentiation is unclear. Differentiation of E14 mouse neurospheres into astrocytes was induced with fetal bovine serum (FBS), Leukemia Inhibitory Factor (LIF), or Ciliary Neurotrophic Factor (CNTF). Cytochemical and enzymatic analyses showed that glycogen is present in FBS- or LIF- but not in CNTF-differentiated astrocytes. Glycogenolysis was induced in FBS- and LIF-differentiated astrocytes but glycogen resynthesis was observed only with FBS. Protein targeting to glycogen mRNA expression appeared with glial fibrillary acidic protein and S100β in FBS and LIF conditions but not with CNTF. These results show that glycogen metabolism constitutes a useful marker of astrocyte differentiation.

scholarly journals Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites

2006 ◽  
Vol 174 (5) ◽  
pp. 715-724 ◽  
Author(s):  
Sandra Angelini ◽  
Diana Boy ◽  
Emile Schiltz ◽  
Hans-Georg Koch
Keyword(s):  
Binding Sites ◽  
Protein Targeting ◽  
Cytoplasmic Membrane ◽  
Signal Recognition Particle ◽  
Membrane Association ◽  
Signal Recognition ◽  
Gtpase Activity ◽  
Signal Recognition Particle Receptor ◽  
Membrane Anchoring ◽  
Lipid Anchor

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRα subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRβ subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY–SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.

scholarly journals Expression and glycogenic effect of glycogen-targeting protein phosphatase 1 regulatory subunit GL in cultured human muscle

2007 ◽  
Vol 405 (1) ◽  
pp. 107-113 ◽  
Author(s):  
Marta Montori-Grau ◽  
Maria Guitart ◽  
Carles Lerin ◽  
Antonio L. Andreu ◽  
Christopher B. Newgard ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Protein Targeting ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Mrna Levels ◽  
Glycogen Accumulation ◽  
Glucose Depletion ◽  
Human Myotubes

Glycogen-targeting PP1 (protein phosphatase 1) subunit GL (coded for by the PPP1R3B gene) is expressed in human, but not rodent, skeletal muscle. Its effects on muscle glycogen metabolism are unknown. We show that GL mRNA levels in primary cultured human myotubes are similar to those in freshly excised muscle, unlike subunits GM (gene PPP1R3A) or PTG (protein targeting to glycogen; gene PPP1R3C), which decrease strikingly. In cultured myotubes, expression of the genes coding for GL, GM and PTG is not regulated by glucose or insulin. Overexpression of GL activates myotube GS (glycogen synthase), glycogenesis in glucose-replete and -depleted cells and glycogen accumulation. Compared with overexpressed GM, GL has a more potent activating effect on glycogenesis, while marked enhancement of their combined action is only observed in glucose-replete cells. GL does not affect GP (glycogen phosphorylase) activity, while co-overexpression with muscle GP impairs GL activation of GS in glucose-replete cells. GL enhances long-term glycogenesis additively to glucose depletion and insulin, although GL does not change the phosphorylation of GSK3 (GS kinase 3) on Ser9 or its upstream regulator kinase Akt/protein kinase B on Ser473, nor its response to insulin. In conclusion, in cultured human myotubes, the GL gene is expressed as in muscle tissue and is unresponsive to glucose or insulin, as are GM and PTG genes. GL activates GS regardless of glucose, does not regulate GP and stimulates glycogenesis in combination with insulin and glucose depletion.

scholarly journals Central Role for Protein Targeting to Glycogen in the Maintenance of Cellular Glycogen Stores in 3T3-L1 Adipocytes

2006 ◽  
Vol 26 (1) ◽  
pp. 334-342 ◽  
Author(s):  
Cynthia C. Greenberg ◽  
Arpad M. Danos ◽  
Matthew J. Brady
Keyword(s):  
Protein Targeting ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Critical Role ◽  
Glycogen Metabolism ◽  
Metabolizing Enzymes ◽  
Protein Levels ◽  
Extracellular Glucose ◽  
Interfering Rna ◽  
Glycogen Stores

ABSTRACT Overexpression of the protein phosphatase 1 (PP1) subunit protein targeting to glycogen (PTG) markedly enhances cellular glycogen levels. In order to disrupt the endogenous PTG-PP1 complex, small interfering RNA (siRNA) constructs against PTG were identified. Infection of 3T3-L1 adipocytes with PTG siRNA adenovirus decreased PTG mRNA and protein levels by >90%. In parallel, PTG reduction resulted in a >85% decrease in glycogen levels 4 days after infection, supporting a critical role for PTG in glycogen metabolism. Total PP1, glycogen synthase, and GLUT4 levels, as well as insulin-stimulated signaling cascades, were unaffected. However, PTG knockdown reduced glycogen-targeted PP1 protein levels, corresponding to decreased cellular glycogen synthase- and phosphorylase-directed PP1 activity. Interestingly, GLUT1 levels and acute insulin-stimulated glycogen synthesis rates were increased two- to threefold, and glycogen synthase activation in the presence of extracellular glucose was maintained. In contrast, glycogenolysis rates were markedly increased, suggesting that PTG primarily acts to suppress glycogen breakdown. Cumulatively, these data indicate that disruption of PTG expression resulted in the uncoupling of PP1 activity from glycogen metabolizing enzymes, the enhancement of glycogenolysis, and a dramatic decrease in cellular glycogen levels. Further, they suggest that reduction of glycogen stores induced cellular compensation by several mechanisms, but ultimately these changes could not overcome the loss of PTG expression.

Differential antagonism of amylin's metabolic and vascular actions with amylin receptor antagonists

1995 ◽  
Vol 73 (7) ◽  
pp. 1025-1029 ◽  
Author(s):  
K. Beaumont ◽  
C. X. Moore ◽  
R. A. Pittner ◽  
K. S. Prickett ◽  
L. S. L. Gaeta ◽  
...  
Keyword(s):  
Nucleus Accumbens ◽  
Binding Sites ◽  
Neuroblastoma Cells ◽  
Glycogen Metabolism ◽  
Calcitonin Gene Related Peptide ◽  
High Affinity ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Rat Nucleus Accumbens ◽  
Potent Antagonist

High affinity amylin binding sites are present in the rat nucleus accumbens. These sites bind [125I]amylin with an affinity of 27 pM and have high affinity for salmon calcitonin (sCT) and moderately high affinity for calcitonin gene related peptide (CGRP). N-terminally truncated peptides were tested for their ability to compete for [125I]amylin binding to these sites and to antagonize the metabolic and vascular actions of amylin. CGRP(8–37), sCT(8–32), and ac-[Asn30,Tyr32]sCT(8–32) (AC187) inhibited [125I]amylin binding to rat nucleus accumbens. Order of potency at inhibiting amylin binding (AC187 > sCT(8–32) > CGRP(8–37)) differed from the order of potency at inhibiting [125I]CGRP binding to SK-N-MC neuroblastoma cells (CGRP(8–37) > AC187 > sCT(8–32)). AC187 was the most potent antagonist of amylin's effects on isolated rat soleus muscle glycogen metabolism, and it was more effective than either sCT(8–32) or CGRP(8–37) at reducing amylin-stimulated hyperlactemia in rats. In contrast, CGRP(8–37) was the most potent peptide at antagonizing amylin-induced hypotension in rats. Amylin's hypotensive actions appear to be mediated by a weak action at CGRP receptors, while its metabolic actions are mediated by receptors with a distinct antagonist profile. AC187 is a potent antagonist of amylin binding sites in nucleus accumbens and of amylin's metabolic actions.Key words: amylin, calcitonin gene related peptide, diabetes, skeletal muscle, peptide receptors.

scholarly journals ROLE OF THE SARCOPLASMIC RETICULUM IN GLYCOGEN METABOLISM

1972 ◽  
Vol 54 (2) ◽  
pp. 206-224 ◽  
Author(s):  
Jean-Claude Wanson ◽  
Pierre Drochmans
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Microsomal Fraction ◽  
Glycogen Metabolism ◽  
Phosphorylase Kinase ◽  
Synthetase Activity ◽  
Polysaccharide Synthesis ◽  
Glycogen Particles ◽  
Differential Precipitation

Sarcoplasmic vesicles and ß-glycogen particles 30–40 mµ in diameter were isolated from perfused rabbit skeletal muscle by the differential precipitation-centrifugation method. This microsomal fraction was subjected to zonal centrifugation on buffered sucrose gradients, in a B XIV Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 mµ, proteins, glycogen, and enzymatic activities (phosphorylase b kinase, phosphorylase b, and glycogen synthetase) were performed. Whereas the entire synthetase activity was found combined with the glycogen particles, 39% of phosphorylase and 53% of phosphorylase b kinase activities, present in the microsomal fraction, were recovered in the purified vesicular fraction (d = 1.175). This latter fraction consists of vesicles, derived from the sarcoplasmic reticulum, and of small particles 10–20 mµ in diameter attached to the outer surface of the membranes. These particles disappear after α-amylase treatment. Incubation of the sarcovesicular fraction with 14C-labeled glucose-1-phosphate confirms the localization of a polysaccharide synthesis at the level of the membranes. "Flash activation" of phosphorylase b, i.e. Ca "activation" of phosphorylase kinase followed by a conversion of phosphorylase b into a, was demonstrated in the purified sarcovesicular fraction. Moreover, the active enzymatic sites were detected on the membranes by electron microscopy. The presence of binding sites between the membranes of the sarcoplasmic vesicles and a glycogen-enzyme complex suggests that this association plays a role in the glycogenolysis during muscle contraction.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

Sites of Insulin Action Using Ferritin Labeling

1971 ◽  
Vol 29 ◽  
pp. 540-541
Author(s):  
Burton B. Silver ◽  
Ronald S. Nelson
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Anterior Pituitary ◽  
Insulin Action ◽  
Diabetic Rats ◽  
Insulin Antibody ◽  
Fat Pad ◽  
Target Organs ◽  
Uranium Salts ◽  
Sugar Levels

Some investigators feel that insulin does not enter cells but exerts its influence in some manner on the cell surface. Ferritin labeling of insulin and insulin antibody was used to determine if binding sites of insulin to specific target organs could be seen with electron microscopy.Alloxanized rats were considered diabetic if blood sugar levels were in excess of 300 mg %. Test reagents included ferritin, ferritin labeled insulin, and ferritin labeled insulin antibody. Target organs examined were were diaphragm, kidney, gastrocnemius, fat pad, liver and anterior pituitary. Reagents were administered through the left common carotid. Survival time was at least one hour in test animals. Tissue incubation studies were also done in normal as well as diabetic rats. Specimens were fixed in gluteraldehyde and osmium followed by staining with lead and uranium salts. Some tissues were not stained.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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