Project description:This a model from the article: Dynamics of muscle glycogenolysis modeled with pH time course computation and pH-dependent reaction equilibria and enzyme kinetics. Vinnakota K, Kemp ML, Kushmerick MJ. Biophys J 2006 Aug 15;91(4):1264-87 16617075 , Abstract: Cellular metabolites are moieties defined by their specific binding constants to H+, Mg2+, and K+ or anions without ligands. As a consequence, every biochemical reaction in the cytoplasm has an associated proton stoichiometry that is generally noninteger- and pH-dependent. Therefore, with metabolic flux, pH is altered in a medium with finite buffer capacity. Apparent equilibrium constants and maximum enzyme velocities, which are functions of pH, are also altered. We augmented an earlier mathematical model of skeletal muscle glycogenolysis with pH-dependent enzyme kinetics and reaction equilibria to compute the time course of pH changes. Analysis shows that kinetics and final equilibrium states of the closed system are highly constrained by the pH-dependent parameters. This kinetic model of glycogenolysis, coupled to creatine kinase and adenylate kinase, simulated published experiments made with a cell-free enzyme mixture to reconstitute the network and to synthesize PCr and lactate in vitro. Using the enzyme kinetic and thermodynamic data in the literature, the simulations required minimal adjustments of parameters to describe the data. These results show that incorporation of appropriate physical chemistry of the reactions with accurate kinetic modeling gives a reasonable simulation of experimental data and is necessary for a physically correct representation of the metabolic network. The approach is general for modeling metabolic networks beyond the specific pathway and conditions presented here. This model was taken from the CellML repository and automatically converted to SBML. The original model was: Vinnakota K, Kemp ML, Kushmerick MJ. (2006) - version=1.0 The original CellML model was created by: Geoffrey Nunns gnunns1@jhu.edu The University of Auckland This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2011 The BioModels.net Team. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information. In summary, you are entitled to use this encoded model in absolutely any manner you deem suitable, verbatim, or with modification, alone or embedded it in a larger context, redistribute it, commercially or not, in a restricted way or not.. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

Project description:This a model from the article: Common phenotype of resting mouse extensor digitorum longus and soleus muscles: equal ATPase and glycolytic flux during transient anoxia. Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 2010 Jun 1;588(Pt 11):1961-83 20308252 , Abstract: Rates of ATPase and glycolysis are several times faster in actively contracting mouse extensor digitorum longus muscle (EDL) than soleus (SOL), but we find these rates are not distinguishable at rest. We used a transient anoxic perturbation of steady state energy balance to decrease phosphocreatine (PCr) reversibly and to measure the rates of ATPase and of lactate production without muscle activation or contraction. The rate of glycolytic ATP synthesis is less than the ATPase rate, accounting for the continual PCr decrease during anoxia in both muscles. We fitted a mathematical model validated with properties of enzymes and solutes measured in vitro and appropriate for the transient perturbation of these muscles to experimental data to test whether the model accounts for the results. Simulations showed equal rates of ATPase and lactate production in both muscles. ATPase controls glycolytic flux by feedback from its products. Adenylate kinase function is critical because a rise in [AMP] is necessary to activate glycogen phosphorylase. ATPase is the primary source of H+ production. The sum of contributions of the 13 reactions of the glycogenolytic and glycolytic network to total proton load is negligible. The stoichiometry of lactate and H+ production is near unity. These results identify a default state of energy metabolism for resting muscle in which there is no difference in the metabolic phenotype of EDL and SOL. Therefore, additional control mechanisms, involving higher ATPase flux and [Ca2+], must exist to explain the well-known difference in glycolytic rates in fast-twitch and slow-twitch muscles in actively contracting muscle. This model was taken from the CellML repository and automatically converted to SBML. The original model was: Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. (2010) - version=1.0 The original CellML model was created by: Geoffrey Nunns gnunns1@jhu.edu The University of Auckland This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2011 The BioModels.net Team. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information. In summary, you are entitled to use this encoded model in absolutely any manner you deem suitable, verbatim, or with modification, alone or embedded it in a larger context, redistribute it, commercially or not, in a restricted way or not.. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

Project description:U2OS osteosarcoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and Raji B-cells in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Bio&Sell), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) at 37°C at 8% or 5% CO2, respectively. Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3’end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.

			Action	DRS
	MODEL1006230077?filename=MODEL1006230077-biopax2.owl	Owl
	MODEL1006230077?filename=MODEL1006230077-biopax3.owl	Owl
	MODEL1006230077?filename=MODEL1006230077.m	Other
	MODEL1006230077?filename=MODEL1006230077.pdf	Pdf
	MODEL1006230077?filename=MODEL1006230077.png	Other

Dataset Information

Vinnakota2006_MuscleGlycogenolysis_ModelA

Dataset's files

Publications

Dynamics of muscle glycogenolysis modeled with pH time course computation and pH-dependent reaction equilibria and enzyme kinetics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets