// DOQCS : http://doqcs.ncbs.res.in/ // Accession Name = cGMP_regulation // Accession Number = 34 // Transcriber = Sudhir Sivakumaran, NCBS // Developer = Sudhir Sivakumaran, NCBS // Species = Bovine and Sprague-Dawley rats; mammalian // Tissue = Pulmonary, cardiac and expressed in Baculovirus infected Sf21(Spodoptera frugiperda) insect cells // Cell Compartment = Cytosol // Notes = Though Corbin JD. et al. Eur J Biochem. (2000) 267(9):2760-7 has been mentioned in the citation, this model has been made with inputs from different literature sources, each of which has been mentioned in the notes sections. This model features hydrolysis of cGMP by bovine PDE, phosphorylation of PDE by bovine lung PKG, and activation of bovine lung PKG by cGMP binding. These mechanisms are known to be involved in cGMP level regulation. Rates have been used from different sources and the model has been tested based on Corbin JD. et al., since their work involved measuring the PDE phosphorylation and PDE activity.
On replicating Figures 2, 3 and 4 from their paper, there is approx 30% difference in results but the qualitative shape of the curves is very similar. This might be due to the fact that the Vmax values were used from different literature sources. This might lead to the discrepancy in the numbers in this model. The values shown in this model are near estimated physiological levels.In order to replicate the Figures more closely, we have run additional simulations with concentration terms changed so as to replicate the experimental conditions exactly. //genesis // kkit Version 11 flat dumpfile // Saved on Thu Dec 8 15:32:05 2005 include kkit {argv 1} FASTDT = 0.0001 SIMDT = 0.001 CONTROLDT = 1 PLOTDT = 1 MAXTIME = 900 TRANSIENT_TIME = 10 VARIABLE_DT_FLAG = 1 DEFAULT_VOL = 1.6667e-21 VERSION = 11.0 setfield /file/modpath value /home2/bhalla/scripts/modules kparms //genesis initdump -version 3 -ignoreorphans 1 simobjdump doqcsinfo filename accessname accesstype transcriber developer \ citation species tissue cellcompartment methodology sources \ model_implementation model_validation x y z simobjdump table input output alloced step_mode stepsize x y z simobjdump xtree path script namemode sizescale simobjdump xcoredraw xmin xmax ymin ymax simobjdump xtext editable simobjdump xgraph xmin xmax ymin 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/kinetics/doqcsinfo 0 db34.g cGMP_regulation network \ "Sudhir Sivakumaran, NCBS" "Sudhir Sivakumaran,NCBS" "citation here" \ "Bovine and Sprague-Dawley rats; mammalian" \ " Pulmonary, cardiac and expressed in Baculovirus infected Sf21(Spodoptera frugiperda) insect cells" \ Cytosol "Quantitative match to experiments, Qualitative" In-house \ "Exact GENESIS implementation" \ "Replicates original data , Approximates original data " 22 -743 0 simundump xgraph /graphs/conc1 0 0 900 0 0.0024221 0 simundump xgraph /graphs/conc2 0 0 900 0 10.023 0 simundump xplot /graphs/conc1/cGMP_PDE*.Co 3 524288 \ "delete_plot.w ; edit_plot.D " 47 0 0 1 simundump xplot /graphs/conc2/5primeGMP.Co 3 524288 \ "delete_plot.w ; edit_plot.D " 22 0 0 1 simundump xgraph /moregraphs/conc3 0 0 1483 0 0.084449 0 simundump xgraph /moregraphs/conc4 0 0 1468 0.058788 0.28079 0 simundump xplot /moregraphs/conc3/cGMP2.PKG.Co 3 524288 \ "delete_plot.w ; edit_plot.D " 24 0 0 1 simundump xplot /moregraphs/conc3/cGMP.Co 3 524288 \ "delete_plot.w ; edit_plot.D " 54 0 0 1 simundump xcoredraw /edit/draw 0 -85 33 -764 -741 simundump xtree /edit/draw/tree 0 \ /kinetics/#[],/kinetics/#[]/#[],/kinetics/#[]/#[]/#[][TYPE!=proto],/kinetics/#[]/#[]/#[][TYPE!=linkinfo]/##[] \ "edit_elm.D ; drag_from_edit.w " auto 0.6 simundump xtext /file/notes 0 1 xtextload /file/notes \ "This model includes the cGMP dependent protein kinase, which is one" \ "of the important downstream molecules. This model features phosphorylation" \ "of PDE by cGMP_PKG, and the direct binding of cGMP with PDE and undergoing" \ "hydrolysis to 5primeGMP. Mechanism of cGMP binding to cGK as proposed by " \ "Taylor et al., 2000, JBC, 275(36):28053-28062. Kinetic parameters used" \ "from the literature cited in the model, as well as refs cited in those " \ "papers. Model was tested using Corbin et al., EJBiochem, 2000, 267:2760-2767." \ "Three plots for the phosphorylation pf PDE and PDE assay were replicated. Though" \ "the shape of the curves matched well, numbers could not be matched exactly, which" \ "might be due to the use of Vmax values of PKG and PDE which were from other" \ "literature sources, since Corbin et al., do not say much about the changes in Vmax." \ "Still we could see the 1.5 to 1.7 fold increase in activity as proposed by Corbin et al" \ "and other papers. A couple of inhibitors were used by Corbin et al., in their expts," \ "which we did not use in the model. So by bringing down the concentration of the PKG and PDe" \ "as do the inhibitors, we could match the numbers. But the values shown in this model are" \ "near physiological levels, and to match the plots we have to replicate the experimental" \ "conditions exactly." addmsg /kinetics/GC/NO_bind_sGCfast /kinetics/GC/NO.sGCfast REAC B A addmsg /kinetics/GC/form_6coord /kinetics/GC/NO.sGCfast REAC A B addmsg /kinetics/GC/form_6coord /kinetics/GC/NO.SGC_6coord REAC B A addmsg /kinetics/GC/act_sGCfast /kinetics/GC/NO.SGC_6coord REAC A B addmsg /kinetics/GC/NO_bind_sGCfast /kinetics/GC/sGCfast REAC A B addmsg /kinetics/GC/sGCfast /kinetics/GC/NO_bind_sGCfast SUBSTRATE n addmsg /kinetics/GC/NO.sGCfast /kinetics/GC/NO_bind_sGCfast PRODUCT n addmsg /kinetics/GC/NO /kinetics/GC/NO_bind_sGCfast SUBSTRATE n addmsg /kinetics/GC/NO.sGCfast /kinetics/GC/form_6coord SUBSTRATE n addmsg /kinetics/GC/NO.SGC_6coord /kinetics/GC/form_6coord PRODUCT n addmsg /kinetics/GC/NO.SGC_6coord /kinetics/GC/act_sGCfast SUBSTRATE n addmsg /kinetics/GC/NO.sGC_5coord /kinetics/GC/act_sGCfast PRODUCT n addmsg /kinetics/GC/nonhemebind_int /kinetics/GC/act_sGCslow SUBSTRATE n addmsg /kinetics/GC/NO2.sGC_5coord /kinetics/GC/act_sGCslow PRODUCT n addmsg /kinetics/GC/NO.sGC6coord /kinetics/GC/NObindnonheme SUBSTRATE n addmsg /kinetics/GC/nonhemebind_int /kinetics/GC/NObindnonheme PRODUCT n addmsg /kinetics/GC/NO /kinetics/GC/NObindnonheme SUBSTRATE n addmsg /kinetics/GC/NObindnonheme /kinetics/GC/nonhemebind_int REAC B A addmsg /kinetics/GC/act_sGCslow /kinetics/GC/nonhemebind_int REAC A B addmsg /kinetics/GC/form6coord /kinetics/GC/NO.sGC6coord REAC B A addmsg /kinetics/GC/NObindnonheme /kinetics/GC/NO.sGC6coord REAC A B addmsg /kinetics/GC/NO.sGCslow /kinetics/GC/form6coord SUBSTRATE n addmsg /kinetics/GC/NO.sGC6coord /kinetics/GC/form6coord PRODUCT n addmsg /kinetics/GC/sGCslow /kinetics/GC/NO_bind_sGCslow SUBSTRATE n addmsg /kinetics/GC/NO.sGCslow /kinetics/GC/NO_bind_sGCslow PRODUCT n addmsg /kinetics/GC/NO /kinetics/GC/NO_bind_sGCslow SUBSTRATE n addmsg /kinetics/GC/NO_bind_sGCslow /kinetics/GC/NO.sGCslow REAC B A addmsg /kinetics/GC/form6coord /kinetics/GC/NO.sGCslow REAC A B addmsg /kinetics/GC/NO_bind_sGCslow /kinetics/GC/sGCslow REAC A B addmsg /kinetics/GC/NO_bind_sGCfast /kinetics/GC/NO REAC A B addmsg /kinetics/GC/NO_bind_sGCslow /kinetics/GC/NO REAC A B addmsg /kinetics/GC/NObindnonheme /kinetics/GC/NO REAC A B addmsg /kinetics/GC/NO.sGC_5coord /kinetics/GC/sGCtot SUMTOTAL n nInit addmsg /kinetics/GC/NO2.sGC_5coord /kinetics/GC/sGCtot SUMTOTAL n nInit addmsg /kinetics/GC/sGCtot /kinetics/GC/sGCtot/sGC_act ENZYME n addmsg /kinetics/GC/GTP /kinetics/GC/sGCtot/sGC_act SUBSTRATE n addmsg /kinetics/GC/act_sGCfast /kinetics/GC/NO.sGC_5coord REAC B A addmsg /kinetics/GC/sGC-ppase*/deph_NO_sGC /kinetics/GC/NO.sGC_5coord REAC sA B addmsg /kinetics/GC/phosph_NO.sGC /kinetics/GC/NO.sGC_5coord REAC B A addmsg /kinetics/GC/deph_NO.sGC /kinetics/GC/phosph_NO.sGC SUBSTRATE n addmsg /kinetics/GC/NO.sGC_5coord /kinetics/GC/phosph_NO.sGC PRODUCT n addmsg 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/kinetics/GC/sGC-ppase* REAC eA B addmsg /kinetics/GC/deph_sGC-ppase /kinetics/GC/sGC-ppase* REAC A B addmsg /kinetics/PDE/cGMP2.PKG/sGCppase_act /kinetics/GC/sGC-ppase* MM_PRD pA addmsg /kinetics/GC/sGC-ppase* /kinetics/GC/sGC-ppase*/deph_NO2_sGC ENZYME n addmsg /kinetics/GC/NO2.sGC_5coord /kinetics/GC/sGC-ppase*/deph_NO2_sGC SUBSTRATE n addmsg /kinetics/GC/sGC-ppase* /kinetics/GC/sGC-ppase*/deph_NO_sGC ENZYME n addmsg /kinetics/GC/NO.sGC_5coord /kinetics/GC/sGC-ppase*/deph_NO_sGC SUBSTRATE n addmsg /kinetics/GC/sGC-ppase* /kinetics/GC/deph_sGC-ppase SUBSTRATE n addmsg /kinetics/GC/sGC-ppase /kinetics/GC/deph_sGC-ppase PRODUCT n addmsg /kinetics/PDE/PDE /kinetics/PDE/PDE/PDE_basal ENZYME n addmsg /kinetics/cGMP /kinetics/PDE/PDE/PDE_basal SUBSTRATE n addmsg /kinetics/PDE/cGMPbindcGK /kinetics/PDE/cGK REAC A B addmsg /kinetics/PDE/PDEbind_cGMP /kinetics/PDE/cGMP.PDE REAC B A addmsg /kinetics/PDE/cGMP2.PKG/PKG_act /kinetics/PDE/cGMP.PDE REAC sA B addmsg /kinetics/PDE/Myo_phosphatase/PP1 /kinetics/PDE/cGMP.PDE MM_PRD pA addmsg /kinetics/PDE/cGMP.PDE/cGMP.PDE_basal /kinetics/PDE/cGMP.PDE REAC eA B addmsg /kinetics/PDE/cGMP.PDE /kinetics/PDE/cGMP.PDE/cGMP.PDE_basal ENZYME n addmsg /kinetics/cGMP /kinetics/PDE/cGMP.PDE/cGMP.PDE_basal SUBSTRATE n addmsg /kinetics/PDE/cGMP_PDE*/PDE_active /kinetics/PDE/5primeGMP MM_PRD pA addmsg /kinetics/PDE/PDE/PDE_basal /kinetics/PDE/5primeGMP MM_PRD pA addmsg /kinetics/PDE/cGMP.PDE/cGMP.PDE_basal /kinetics/PDE/5primeGMP MM_PRD pA addmsg /kinetics/PDE/cGMP.PDE /kinetics/PDE/PDEbind_cGMP PRODUCT n addmsg /kinetics/cGMP /kinetics/PDE/PDEbind_cGMP SUBSTRATE n addmsg /kinetics/PDE/PDE /kinetics/PDE/PDEbind_cGMP SUBSTRATE n addmsg /kinetics/cGMP /kinetics/PDE/cGMPbindcGK SUBSTRATE n addmsg /kinetics/PDE/cGK /kinetics/PDE/cGMPbindcGK SUBSTRATE n addmsg /kinetics/PDE/cGMP.PKG /kinetics/PDE/cGMPbindcGK PRODUCT n addmsg /kinetics/PDE/cGMP2.PKG/PKG_act /kinetics/PDE/cGMP_PDE* MM_PRD pA addmsg /kinetics/PDE/Myo_phosphatase/PP1 /kinetics/PDE/cGMP_PDE* REAC sA B addmsg /kinetics/PDE/cGMP_PDE* /kinetics/PDE/cGMP_PDE*/PDE_active ENZYME n addmsg /kinetics/cGMP /kinetics/PDE/cGMP_PDE*/PDE_active SUBSTRATE n addmsg /kinetics/PDE/cGMP.PKG /kinetics/PDE/fast_site_bind SUBSTRATE n addmsg /kinetics/PDE/cGMP2.PKG /kinetics/PDE/fast_site_bind PRODUCT n addmsg /kinetics/cGMP /kinetics/PDE/fast_site_bind SUBSTRATE n addmsg /kinetics/PDE/Myo_phosphatase/PP1 /kinetics/PDE/Myo_phosphatase REAC eA B addmsg /kinetics/PDE/Myo_phosphatase /kinetics/PDE/Myo_phosphatase/PP1 ENZYME n addmsg /kinetics/PDE/cGMP_PDE* /kinetics/PDE/Myo_phosphatase/PP1 SUBSTRATE n addmsg /kinetics/PDE/cGMPbindcGK /kinetics/PDE/cGMP.PKG REAC B A addmsg /kinetics/PDE/fast_site_bind /kinetics/PDE/cGMP.PKG REAC A B addmsg /kinetics/PDE/fast_site_bind /kinetics/PDE/cGMP2.PKG REAC B A addmsg /kinetics/PDE/cGMP2.PKG/PKG_act /kinetics/PDE/cGMP2.PKG REAC eA B addmsg /kinetics/PDE/cGMP2.PKG/sGCppase_act /kinetics/PDE/cGMP2.PKG REAC eA B addmsg /kinetics/PDE/cGMP2.PKG /kinetics/PDE/cGMP2.PKG/PKG_act ENZYME n addmsg /kinetics/PDE/cGMP.PDE /kinetics/PDE/cGMP2.PKG/PKG_act SUBSTRATE n addmsg /kinetics/PDE/cGMP2.PKG /kinetics/PDE/cGMP2.PKG/sGCppase_act ENZYME n addmsg /kinetics/GC/sGC-ppase /kinetics/PDE/cGMP2.PKG/sGCppase_act SUBSTRATE n addmsg /kinetics/PDE/PDEbind_cGMP /kinetics/cGMP REAC A B addmsg /kinetics/PDE/cGMPbindcGK /kinetics/cGMP REAC A B addmsg /kinetics/PDE/fast_site_bind /kinetics/cGMP REAC A B addmsg /kinetics/GC/sGCtot/sGC_act /kinetics/cGMP MM_PRD pA addmsg /kinetics/PDE/cGMP_PDE*/PDE_active /kinetics/cGMP REAC sA B addmsg /kinetics/PDE/PDE/PDE_basal /kinetics/cGMP REAC sA B addmsg /kinetics/PDE/cGMP.PDE/cGMP.PDE_basal /kinetics/cGMP REAC sA B addmsg /kinetics/PDE/cGMP_PDE* /graphs/conc1/cGMP_PDE*.Co PLOT Co *cGMP_PDE*.Co *47 addmsg /kinetics/PDE/5primeGMP /graphs/conc2/5primeGMP.Co PLOT Co *5primeGMP.Co *22 addmsg /kinetics/PDE/cGMP2.PKG /moregraphs/conc3/cGMP2.PKG.Co PLOT Co *cGMP2.PKG.Co *24 addmsg /kinetics/cGMP /moregraphs/conc3/cGMP.Co PLOT Co *cGMP.Co *54 enddump // End of dump call /kinetics/GC/notes LOAD \ "This group features the binding reaction scheme as proposed by" \ "Stone and Marletta,1996, Biochemistry, 35(4):1093-1099. They" \ "report a two step binding step for the activation of sGC by NO." \ "Rates shown in the model are used directly from their experiments" \ "on stopped flow kinetics. " call /kinetics/GC/sGCfast/notes LOAD \ "Stone and Marletta, Biochemistry,35(4), 1996," \ "" \ "They have reported that the binding of sGC with NO occurs through two" \ "phases;a slow process and a fast process. 30% of the sGC binds " \ "quickly to NO; but the rest 70% goes through a slow process which" \ "involves the binding of another NO to an unidentified non-heme " \ "site on the protein, which seems to be not necessarily the same " \ "site used in the initial two step binding to the heme." \ "" \ "Stone and Marletta,1995,34;14668-14674;-- report that the native" \ "heme stoichiometry is 2 per heterodimer. spectrally only one" \ "type of heme is observed, indicating that both hemes are in " \ "similar environment. they conclude that each subunit of the " \ "heterodimer binds 1 equiv of heme at a site conserved between" \ "the two subunits." \ "" \ "Concentration 3 uM assumed on the basis of reported data" \ "(Kuroda et al., J.Neurosci., 2001, 21(15): 5693-5702 ;" \ "Ariano et al., PNAS, 1982, 79:1316-1320)" \ "" \ "" \ "" \ "" call /kinetics/GC/NO_bind_sGCfast/notes LOAD \ "This is the fast binding of NO to sGC, subsequently activating" \ "it, as proposed by Stone and Marletta." \ "" \ "Rates of these binding reactions used directly from Stone and" \ "Marletta,1996, Biochemistry, 35(4):1093-1099, " \ "based on whose paper this model is made. Rates obtained from" \ "stopped flow kinetics detailed in their paper." call /kinetics/GC/form_6coord/notes LOAD \ "Rates used directly from Stone and Marletta,1996,Biochemistry," \ "35(4):1093-1099." call /kinetics/GC/act_sGCfast/notes LOAD \ "Rates used directly from Stone and Marletta,1996,Biochemistry," \ "35(4):1093-1099." call /kinetics/GC/act_sGCslow/notes LOAD \ "Rates used directly from Stone and Marletta,1996, Biochemistry," \ "35(4):1093-1099." call /kinetics/GC/NObindnonheme/notes LOAD \ "This step is the one that differs from the fast reaction scheme," \ "as reported by Stone and Marletta. Here the reaction is dependent " \ "upon the binding of NO to an unidentified non-heme site on the" \ "protein." \ "" \ "Rates used directly from Stone and Marletta,1996, Biochemistry," \ "35(4):1093-1099." call /kinetics/GC/nonhemebind_int/notes LOAD \ "here the new NO has bound to the unidentified non-heme site on" \ "the 6-coordinate complex, and this will convert to the" \ "5-coordinate complex. this binding of a new NO to the non-heme " \ "site on the 6-coordinate complex is characteristic of the slow " \ "binding process of NO to sGC." call /kinetics/GC/NO.sGC6coord/notes LOAD \ "this compoud, a 6-coordinate complex will be bound by another NO" \ "to an unidentified non-heme site in the next reaction step." call /kinetics/GC/form6coord/notes LOAD \ "Rates used directly from Stone and Marletta,1996, Biochemistry," \ "35(4):1093-1099." call /kinetics/GC/NO_bind_sGCslow/notes LOAD \ "This is the slow binding of NO to sGC, as reported by Stone and " \ "Marletta,1996, Biochemistry, 35(4):1093-1099. " \ "The rates shown have been used directly from their data from" \ "stopped flow kinetics." call /kinetics/GC/sGCslow/notes LOAD \ "This is the slow reaction process of the binding of NO to sGC." \ "we can notice the binding of another NO in the third reaction " \ "step to a non-heme site, which then converts to the 5-coordinate" \ "complex. this binding of another NO to an non-heme site is" \ "characteristic of the slow binding process of NO to sGC.(Stone" \ "and Marletta, 1996,35(4);1093-1099)" \ "" \ "Concentration 3 uM assumed on the basis of reported data " \ "(Kuroda et al., J.Neurosci., 2001, 21(15): 5693-5702 ; " \ "Ariano et al., PNAS, 1982, 79:1316-1320.)" call /kinetics/GC/NO/notes LOAD \ "Endogenously produced NO concentrations in the course of" \ "signal transduction processes are < 100 nM. (Varner et al.," \ "Nitric oxide in the nervous system, Academic press, London, UK," \ "pp.191-206.)" call /kinetics/GC/sGCtot/notes LOAD \ "This is the sumtotal of the sGC activated via two binding " \ "mecanisms as reported by Stone and Marletta, 1996, Biochemistry," \ "35(4):1093-1099." call /kinetics/GC/sGCtot/sGC_act/notes LOAD \ "The range of estimates found in the literature are:" \ "Km -> 40 - 150 uM (without NO)" \ " 20 - 40 uM (with NO)" \ "Vmax -> 10 - 100 nmol/mg/min (wihtout NO)" \ " 10 - 40 umol/mg/min (with NO)." \ "----- thru personal correspondence from T. Bellamy, Wolfson " \ "Ins. for Biomedical Sciences, UK." \ "NO increases the Vmax of sGC by 100-200 fold, and it has been " \ "proposed that this activation occurs subsequent to the binding " \ "of NO toa heme moiety on the enzyme. (Stone and Marletta,1995," \ "Biochemistry,34:14668-14674). " call /kinetics/GC/NO.sGC_5coord/notes LOAD \ "This is the active sGC, a 5-coordinate complex formed after a " \ "series of reaction steps, involving the formation of a " \ "6 coordinate complex which converted to a 5-coordinate complex." call /kinetics/GC/phosph_NO.sGC/notes LOAD \ "Rates fixed based on teh ratio of active and inactive forms." call /kinetics/GC/NO2.sGC_5coord/notes LOAD \ "This is the 5-coordinate complex of the enzyme sGC. This is" \ "also the active form of the enzyme." call /kinetics/GC/phosph_NO2.sGC/notes LOAD \ "Rates fixed based on the ratio of active and inactive forms." call /kinetics/GC/sGC-ppase*/deph_NO2_sGC/notes LOAD \ "similar to deph_NO_sGC.." call /kinetics/GC/sGC-ppase*/deph_NO_sGC/notes LOAD \ "Km and Vmax used from Ferrero et al., J Neurochem, 2000, 75:" \ "2029-2039, and other refs cited in their paper. Though there" \ "are no plots in this paper, the rates look ok when the model " \ "is being run. Still this part can be improved further based " \ "on more experimental observations, since at the moment, there" \ "is not much information on the rates and kinetic details" \ "of the phosphatase mediated sGC regulation." call /kinetics/GC/deph_sGC-ppase/notes LOAD \ "Rates fixed from the ratio of the inactive and active (phos) " \ "form of sGC-ppase." call /kinetics/PDE/notes LOAD \ "This model features the hydrolysis of cGMP by PDE. (The " \ "PDE taken into consideration is PDE 5, a hgihly cGMP specific " \ "PDE, and is considered to be the main enzyme responsible for " \ "terminating the action of cGMP generated following the release " \ "of NO from Nitrergic nerves, or from vascular endothelial cells." \ "(Gibson, Eur J Biochem,2001,411:1-10). PDE is phosphorylated by" \ "PKG. (Corbin et al., Eur J Biochem,2000,267:2760-2767). And" \ "occupation of the allosteric binding sites on PDE by cGMP is " \ "necessary for phosphorylation by PKG. (Gibson, Eur J Biochem," \ "2001,411:1-10)." \ "Three plots in the paper by Corbin et al., were replicated with this" \ "model. Though the shape of the curves matched well, the numbers could not be" \ "matched due to the differences in Vmax values used from different literature " \ "sources apart from Corbin et al. Various inhibitors were used by Corbin et al., " \ "in their expts and so to match the numbers in the plots we have to replicate the " \ "experimental conditions exactly. So by bringing down the concentration of PKG and PDE" \ "we could match the numbers as well. But the values shown in this model are near " \ "physiological levels, as thats what we intend to do basically -- Building models of" \ "signaling pathways whose behaviour is similar to what happens invivo." call /kinetics/PDE/PDE/notes LOAD \ "Phophosdiesterase. Degrades cGMP to 5primeGMP. " \ "Concentration used by Kuroda et al.,(J Neurosci, 2001,21(15):" \ "5693-5702) and initially from Kotera et al., " \ "(1997, Eur J Biochem, 249:434-442). PDE 5 is highly specific " \ "for cGMP and is considered to be the main enzyme responsible " \ "for terminating the action of cGMP generated following the release" \ "of NO from nitrergic nerves, or from vascular endothelial cells." \ "(Gibson,Eur J Pharmacol, 2001, 411:1-10). Occupation of the " \ "allosteric binding sites by cGMP is neessary for phosphorylation" \ "by PKG. In Corbinet al., EJBiochem, 2000, 267:2760-2767, " \ "Phosphorylation of PDE and PDE activity are measured, but" \ "as mentioned, due to the different sources of Vmax values" \ "the numbers could not be matched exactly with respect to the plots" \ "in the paper, and they were either more or less by a factor" \ "of two. To match the numbers exactly we have to replicate the" \ "experimental conditions exactly. " \ "" call /kinetics/PDE/cGK/notes LOAD \ "cGMP dependent protein kinase (also cGKI and cGKII)" \ "They are both present in the brain. While cGKI is selectively" \ "present only in the Purkinje cells of the cerebellum, cGKII" \ "is widely expressed in the brain tissue and may be a neuronal" \ "target of cGMP. (El-Husseini et al., J Neurochem, 1995;64:2814;" \ "Hofmann et al., 2000, J Cell Sci, 113:1671-1676; Lohmann et al.," \ "1997, Trends Biochem Sci, 22:307-312). PKG is a dimer, though " \ "the function of dimerization is not known, with some reports" \ "indicating that dimerization may not play a vital role in the" \ "activity of the enzyme. This is the inactive form, which on being " \ "bound by cGMP becomes active. The binding sites are described by their" \ "distinct rates of dissociation of bound cyclic nucleotide " \ "(fast and slow). - Taylor et al., 2000,JBC, 275(36):28053-28062. " \ "Intracellular concentration of PKG estimated to be present in " \ "vascular smooth muscle cells is around 0.3 - 0.5 uM. " \ "(Corbin et al., 2000, Eur J Biochem,267:2760-2767) " \ "" call /kinetics/PDE/cGMP.PDE/notes LOAD \ "cGMP bound PDE. Binding of cGMP to the allosteric cGMP-binding" \ "sites has been reported to be required for phosphorylation by" \ "cGMP-dep protein Kinase, and the elements contributing to the" \ "dimerization of this protein are located in or near the " \ "allosteric cGMP-binding sites, though the function of " \ "dimerization is unknown. " \ "(Fink et al., JBC, 1999, 274(49):34613-34620)" \ " " call /kinetics/PDE/5primeGMP/notes LOAD \ "5primeGMP - The hydrolysed product from cGMP, by the catalytic" \ "activity of PDE's. In this case the cGMP specific PDE, PDE5." call /kinetics/PDE/PDEbind_cGMP/notes LOAD \ "Kd ~1.3 uM. " \ "(Turko et al., JBC, 1996, 271(36):22240-22244, and " \ "Corbin et al., Eur J Biochem, 2000,267:2760-2767)" \ "" call /kinetics/PDE/cGMPbindcGK/notes LOAD \ "Kinase activation in both the isoforms of cGK depends on " \ "cyclic nucleotide occupation of the two cyclic nucleotide " \ "binding sites in the regulatory domain. This event is supposed " \ "to reduce the affinity of the auto-inhibition region of the " \ "regulatory domain for the catalytic domain. Investigations" \ "revealed that cGMP binds to a slowly dissociating cyclic " \ "nucleotide binding site and induces a conformational change" \ "resulting in a partially active kinase. Subsequent occupation" \ "of the second, rapid dissociation site imparts additional " \ "conformational change until it forms the elongated shape " \ "that is reported to be associated with the fully active enzyme." \ "(Taylor et al., 2000, JBC, 275(36):28053-28062)" \ "Dissociation rates for cGKII binding sites from " \ "Taylor et al., 2000, and other refs cited in their paper." \ "cGMP dissociation from slow site -- 8.1/s (Smith et al., " \ "JBC,1995, 271(34):20756-20762)" \ "" call /kinetics/PDE/cGMP_PDE*/notes LOAD \ "Phosphorylated PDE. Phosphorylated by PKG, which requires cGMP" \ "to be bound to the allosteric binding sites on PDE. Hence there " \ "are more than one molecule of cGMP bound to the active enzyme" \ "complex, since initially, cGMP binding to the allosteric " \ "binding sites is necessary for phosphorylation of PDE by PKG." \ "" call /kinetics/PDE/cGMP_PDE*/PDE_active/notes LOAD \ "In this cGMP_PDE* enzyme complex, there are more than one cGMP" \ "molecule bound, since initially, cGMP binding to the allosteric" \ "binding sites is essential for phosphorylation by PKG. " \ "Vmax initially from Turko et al., 1998, Biochem J, " \ "329:505-510 and Kuroda et al., 2001, J Neurosci,21(15):5693-5702." \ "PDE has a high catalytic rate for cGMP hydrolysis. " \ "Km values reported are ~1-2 uM in various studies, including" \ "studies with purified enzyme preparations.(Fink et al., JBC," \ "1999, 274(49):34613-34620 and cited refs in their paper)." \ "Value used here from Mehats et al., Trends in Endocrinology &" \ "Metabolism, 2002, 13(1):29-35. Values similar to those used " \ "by Kuroda et al.,J Neurosci, 2001, 21(15):5693-5702." \ "Km for cGMP decreased from 0.98 to 0.58 uM and Vmax was " \ "reported to be slightly increased by phosphorylation." \ "(Corbin et al., Eur J Biochem, 2000)" call /kinetics/PDE/Myo_phosphatase/notes LOAD \ "Rybalkin et al., JBC, 2002, 277(5):3310-3317." \ "Myosin phosphatase reported to be involved in " \ "dephosphorylation of PDE5." \ "Rates from Rybalkin et al., 2002." call /kinetics/PDE/cGMP.PKG/notes LOAD \ "cGK bound by cGMP to the amino terminal slow site, which " \ "is subsequently bound to the fast site in the next step by " \ "another cGMP molecule. (Taylor et al., JBC, 2000, 275(36):" \ "28053-28062). Rates from Taylor et al., 2000." \ "" call /kinetics/PDE/cGMP2.PKG/notes LOAD \ "Phosphorylated cGMP-dependent protein kinase. cGMP binding to" \ "the inactive cGK activates it, making it participate in " \ "further downstream regulatory processes, mostly through " \ "phosphorylation regulation of its substrates. Here it " \ "phosphorylates PDE, which hydrolyses cGMP to 5primeGMP, thus" \ "maintaining the intracellular nucleotide levels. The activity " \ "of PDE is supposed to be enhanced following phosphorylation by" \ "PKG." call /kinetics/PDE/cGMP2.PKG/PKG_act/notes LOAD \ "Phosphorylation of PDE actually increases the enzyme activity." \ "And occupation of the allosteric binding sites by cGMP is " \ "necessary for phosphorylation by PKG. Phosphorylation following" \ "the occupation of the allosteric sites by cGMP." \ "(Gibson, Eur J Pharmacol,2001,411:1-10, Corbin et al., 2000," \ "Eur J Biochem, 267:2760-2767). Phosphorylation resulted in " \ "increases in phosphate content up to 0.6 mol per PDE5 subunit." \ "(Corbin et al., 2000), and since PKG is known to catalyze" \ "autophosphorylation, it was found to incorporate 2 mol per PDE5" \ "subunit in 60 mins." call /kinetics/cGMP/notes LOAD \ "cGMP takes part in various reactions downstream. One of the " \ "interesting schemes are the binding of cGMP to the allosteric " \ "cGMP binding sites, which is supposed to be essential for the" \ "phosphorylation of PDE by PKG. So in the cGMP_PDE* enzyme " \ "complex, there is more then one cGMP molecule bound. " call /kinetics/doqcsinfo/notes LOAD \ "Though Corbin JD. et al. Eur J Biochem. (2000) 267(9):2760-7 has been mentioned in the citation, this model has been made with inputs from different literature sources, each of which has been mentioned in the notes sections. This model features hydrolysis of cGMP by bovine PDE, phosphorylation of PDE by bovine lung PKG, and activation of bovine lung PKG by cGMP binding. These mechanisms are known to be involved in cGMP level regulation. Rates have been used from different sources and the model has been tested based on Corbin JD. et al., since their work involved measuring the PDE phosphorylation and PDE activity.
On replicating Figures 2, 3 and 4 from their paper, there is approx 30% difference in results but the qualitative shape of the curves is very similar. This might be due to the fact that the Vmax values were used from different literature sources. This might lead to the discrepancy in the numbers in this model. The values shown in this model are near estimated physiological levels.In order to replicate the Figures more closely, we have run additional simulations with concentration terms changed so as to replicate the experimental conditions exactly." complete_loading