 |  |  Ras |
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| | Name | Initial Conc. (uM) | Volume (fL) | Buffered | | 1 | AA | 6.12 | 1000 | No | | | Arachidonic Acid. This messenger diffuses through membranes as well as cytosolically, has been suggested as a possible retrograde messenger at synapses. | | 2 | Ca | 0.08 | 1000 | Yes | | | This calcium pool is treated as being buffered to a steady 0.08 uM, which is the resting level. | | 3 | DAG | 11.661 | 1000 | Yes | | | 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. | | 4 | 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 | | 5 | MKP-2 | 0.0024 | 1000 | No | | | MKP2 is modeled to act as a relatively steady, unregulated phosphatase for controlling MAPK activity. From Brondello et al JBC 272(2):1368-1376 (1997), the blockage of MKP-1 induction increases MAPK activity by no more than 2x. So this phosphatase will play the steady role and the fully stimulated MKP-1 can come up to the level of this steady level. From Chu et al 1995 JBC 271(11):6497-6501 it looks like both MKP-1 and MKP-2 have similar activities in dephosphorylating ERK2. So I use the same enzymatic rates for both. 31 Jan 2002: For the purposes of making a bistable model without the complications of MKP-1 induction, I simply set the initial conc of MKP-2 up by 0.0004 uM which was the starting level of MKP-1. | | 6 | 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. | | 7 | 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. | | 8 | Shc*.Sos.Grb2 | 0 | 1000 | No | | | This three-way complex is one of the main GEFs for activating Ras. | | 9 | 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. |
Summed Molecule List | | Target | Inputs | | 1 |
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. |
Pathway Details Molecule List Enzyme List Reaction List
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