| | Name | Initial Conc. (uM) | Volume (fL) | Buffered |
| 1 | AA | 0 | 1000 | No |
| | Arachidonic Acid. This messenger diffuses through membranes as well as cytosolically, has been suggested as a possible retrograde messenger at synapses. |
| 2 | BetaGamma | 0 | 1000 | No |
| | The betagamma subunits of Gq. This is an approximation to the possible combinations of betagamma subunits. Here they are all treated as a single pool. |
| 3 | Ca | 0.08 | 1000 | No |
| | This calcium pool is treated as being buffered to a steady 0.08 uM, which is the resting level. |
| 4 | CaM(Ca)n-CaNAB | 0 | 1000 | No |
| | This pool sums the levels of the CaM.Ca2.CaNAB, CaM.Ca3.CaNAB and CaM.Ca4.CaNAB pools. |
| 5 | CaM-Ca3 | 0 | 1000 | No |
| | The TR1 end now begins to bind Ca. This form has 2 Ca's on the TR2 end, and one on the TR1. |
| 6 | CaM-Ca4 | 0 | 1000 | No |
| | The four-calcium-bound form of CaM. It is the active form for most reactions. |
| 7 | CaM-TR2-Ca2 | 0 | 1000 | No |
| | This is the intermediate where the TR2 end (the high-affinity end) has bound the Ca but the TR1 end has not. |
| 8 | cAMP | 0 | 1000 | No |
| | |
| 9 | CaNAB-Ca4 | 0 | 1000 | No |
| | Four calciums bound to CaN. This has an activity in absence of CaM. Perrino et al 1992 JBC 267(22):15965-15969 |
| 10 | Ca_intracell | 0.08 | 1000 | No |
| | This is the pool representing intracellular calcium. Resting levels are around 80 nM, but this is subject to all sorts of influxes and pumps. |
| 11 | Ca_stim | 0 | 1000 | No |
| | |
| 12 | DAG | 0 | 1000 | No |
| | Baseline in model is 11.661 uM. DAG is pretty nasty to estimate. In this model we just hold it fixed at this baseline level. Data sources are many and varied and sometimes difficult to reconcile. Welsh and Cabot 1987 JCB 35:231-245: DAG degradation Bocckino et al JBC 260(26):14201-14207: hepatocytes stim with vasopressin: 190 uM. Bocckino et al 1987 JBC 262(31):15309-15315: DAG rises from 70 to 200 ng/mg wet weight, approx 150 to 450 uM. Prescott and Majerus 1983 JBC 258:764-769: Platelets: 6 uM. Also see Rittenhouse-Simmons 1979 J Clin Invest 63. Sano et al JBC 258(3):2010-2013: Report a nearly 10 fold rise. Habenicht et al 1981 JBC 256(23)12329-12335: 3T3 cells with PDGF stim: 27 uM Cornell and Vance 1987 BBA 919:23-36: 10x rise from 10 to 100 uM. Summary: I see much lower rises in my PLC models, but the baseline could be anywhere from 5 to 100 uM. I have chosen about 11 uM based on the stimulus -response characteristics from the Schaechter and Benowitz paper and the Shinomura et al papers. |
| 13 | G*GDP | 0 | 1000 | No |
| | This should correctly be called GDP.G_alpha. The name is preserved for backward compatibility reasons. |
| 14 | G*GTP | 0 | 1000 | No |
| | Activated G protein. Berstein et al indicate that about 20-40% of the total Gq alpha should bind GTP at steady stimulus. |
| 15 | Glu | 0 | 1000 | No |
| | Varying the amount of (steady state) glu between .01 uM and up, the final amount of G*GTP complex does not change much. This means that the system should be reasonably robust wr to the amount of glu in the synaptic cleft. It would be nice to know how fast it is removed. Schoepp et al 1990 TIPS 11:508-515 give a range of Glu EC50 from rat brain in the range 120 to 1000 uM. Nicoletti 1986 PNAS 83:1931-1935 and Schoepp and Johnson 1989 J Neurochem 53:1865-1870 give an off time of at least 30 sec. |
| 16 | Gs-alpha | 0 | 1000 | No |
| | This is actualy GTP.Gs_alpha, the active form of Gs. Resting Gs-alpha is nearly zero. |
| 17 | IP3 | 0.73 | 1000 | No |
| | Peak IP3 is ~ 15 uM, basal ~ 0.2 uM. |
| 18 | MAPK* | 0 | 1000 | No |
| | This molecule is phosphorylated on both the tyr and thr residues and is active: Seger et al 1992 JBC 267(20):14373 The rate consts are from two sources: Combine Sanghera et al JBC 265(1) :52-57 with Nemenoff et al JBC 93 pp 1960 to get k3 = 10, k2 = 40, k1 = 3.25e-6 |
| 19 | MKP-1 | 0.0032 | 1000 | No |
| | MKP-1 dephosphorylates and inactivates MAPK in vivo: Sun et al Cell 75 487-493 1993. See Charles et al PNAS 90:5292-5296 1993 and Charles et al Oncogene 7 187-190 for half-life of MKP1/3CH is 40 min. 80% deph of MAPK in 20 min The protein is 40 KDa. The levels are MKP-1 are highly variable, as it is induced depending on MAPK activity. This selected value is well below its induced peak, but sufficiently high so that MAPK will not go into a runaway activation state. |
| 20 | PIP2 | 10 | 1000 | Yes |
| | PIP2 is a bit troublesome in this model. Its level is well below what it should be based on more recent data. This value is kept in this model to correspond to the Km used in the enzymes. A scale factor of 5-10 in both terms would cancel out but improve the parameter estimate. |
| 21 | PKA-active | 0 | 1000 | No |
| | The free catalytic subunit. |
| 22 | PKC-active | 0.02 | 1000 | No |
| | This is the total active PKC. It is the sum of the respective activities of PKC-basal* PKC-Ca-memb* PKC-DAG-memb* PKC-Ca-AA* PKC-DAG-AA* PKC-AA* I treat PKC here in a two-state manner: Either it is in an active state (any one of the above list) or it is inactive. No matter what combination of stimuli activate the PKC, I treat it as having the same activity. The scaling comes in through the relative amounts of PKC which bind to the respecive stimuli. The justification for this is the mode of action of PKC, which like most Ser/Thr kinases has a kinase domain normally bound to and blocked by a regulatory domain. I assume that all the activators simply free up the kinase domain. A more general model would incorporate a different enzyme activity for each combination of activating inputs, as well as for each substrate. The current model seems to be a decent and much simpler approximation for the available data. One caveat of this way of representing PKC is that the summation procedure assumes that PKC does not saturate with its substrates. If this assumption fails, then the contributing PKC complexes would experience changes in availability which would affect their balance. Given the relatively low percentage of PKC usually activated, and its high throughput as an enzyme, this is a safe assumption under physiological conditions. |
| 23 | PP1-active | 1.8 | 1000 | No |
| | Cohen et al Meth Enz 159 390-408 is main source of info concentration of enzyme = 1.8 uM |
| 24 | PP2A | 0.12 | 1000 | No |
| | Protein phosphatase 2A dephosphorylates the inibitory subunit. This is treated as a basal level of dephosphorylation. More specific and regulated dephosphorylation occurs through CaN, calcineurin. Cohen 1989: Ann rev Biochem 58:453-508 |
| 25 | PPhosphatase2A | 0.224 | 1000 | No |
| | Refs: Pato et al Biochem J 293:35-41(93); CoInit values span a range depending on source. Pato et al 1993 Biochem J 293:35-41 and Cohen et al 1988 Meth Enz 159:390-408 estimate 80 nM from muscle Zolneierowicz et al 1994 Biochem 33:11858-11867 report levels of 0.4 uM again from muscle, but expression is also strong in brain. Our estimate of 0.224 is between these two. There are many substrates for PP2A in this model, so I put the enzyme rate calculations here: Takai&Mieskes Biochem J 275:233-239 have mol wt 36 KDa. They report Vmax of 119 umol/min/mg i.e. 125/sec for k3 for pNPP substrate, Km of 16 mM. This is obviously unreasonable for protein substrates. For chicken gizzard myosin light chan, we have Vmax = 13 umol/min/mg or about k3 = 14/sec. Pato et al 1993 Biochem J 293:35-41 report caldesmon: Km = 2.2 uM, Vmax = 0.24 umol/min/mg. They do not think caldesmon is a good substrate. Calponin: Km = 14.3, Vmax = 5. Our values approximate these. |
| 26 | Shc*.Sos.Grb2 | 0 | 1000 | No |
| | This three-way complex is one of the main GEFs for activating Ras. |
| 27 | synapse | 0 | 1000 | No |
| | A pool representing the presynaptic terminal and release of glutamate. It is controlled by the temporal pattern of the synaptic input. |
| 28 | temp-PIP2 | 2.5 | 1000 | Yes |
| | This is a steady PIP2 input to PLA2. The sensitivity of PLA2 to PIP2 discussed below does not match with the reported free levels which are used by the phosphlipase Cs. My understanding is that there may be different pools of PIP2 available for stimulating PLA2 as opposed to being substrates for PLCs. For that reason I have given this PIP2 pool a separate identity. As it is a steady input this is not a problem in this model. Majerus et al Cell 37:701-703 report a brain concentration of 0.1 - 0.2 mole % Majerus et al Science 234:1519-1526 report a huge range of concentrations: from 1 to 10% of PI content, which is in turn 2-8% of cell lipid. This gives 2e-4 to 8e-3 of cell lipid. In concentrations in total volume of cell (a somewhat strange number given the compartmental considerations) this comes to anywhere from 4 uM to 200 uM. PLA2 is stim 7x by PIP2 (Leslie and Channon BBA 1045:261(1990) Leslie and Channon say PIP2 is present at 0.1 - 0.2mol% range in membs, so I'll use a value at the lower end of the scale for basal PIP2. |
| | Target | Inputs |
| 1 |
Ca | Ca_stim
Ca_intracell
|
This calcium pool is treated as being buffered to a steady 0.08 uM, which is the resting level. | | 2 |
CaM(Ca)n-CaNAB | CaMCa4-CaNAB
CaMCa3-CaNAB
CaMCa2-CANAB
|
This pool sums the levels of the CaM.Ca2.CaNAB, CaM.Ca3.CaNAB and CaM.Ca4.CaNAB pools. | | 3 |
PKC-active | PKC-DAG-AA*
PKC-Ca-memb*
PKC-Ca-AA*
PKC-DAG-memb*
PKC-basal*
PKC-AA*
|
This is the total active PKC. It is the sum of the respective activities of PKC-basal* PKC-Ca-memb* PKC-DAG-memb* PKC-Ca-AA* PKC-DAG-AA* PKC-AA* I treat PKC here in a two-state manner: Either it is in an active state (any one of the above list) or it is inactive. No matter what combination of stimuli activate the PKC, I treat it as having the same activity. The scaling comes in through the relative amounts of PKC which bind to the respecive stimuli. The justification for this is the mode of action of PKC, which like most Ser/Thr kinases has a kinase domain normally bound to and blocked by a regulatory domain. I assume that all the activators simply free up the kinase domain. A more general model would incorporate a different enzyme activity for each combination of activating inputs, as well as for each substrate. The current model seems to be a decent and much simpler approximation for the available data. One caveat of this way of representing PKC is that the summation procedure assumes that PKC does not saturate with its substrates. If this assumption fails, then the contributing PKC complexes would experience changes in availability which would affect their balance. Given the relatively low percentage of PKC usually activated, and its high throughput as an enzyme, this is a safe assumption under physiological conditions. |
