| | Name | Initial Conc. (uM) | Volume (fL) | Buffered |
| 1 | ADP | 0 | 0.0016667 | No |
| | ADP obtained as a result of phosporylation of SERCA2a with ATP. |
| 2 | Ca | 0.268 | 0.0016667 | Yes |
| | For simulation of EP formation and Pi liberation time couse at various [ca2+] the concentrations were obtained from the literature source Mahaney et al. Biophysical Journal 78(3) 1306-1323 ******************************************************************** In case of EP decomposition after phosphorylation with 1uM ATP for 30s at 0 deg centigrade. At 30s 5mM EGTA (final conc) was added to the reaction mixture to remove all traces of ionized Ca2+ form the reaction mixture and thus to reduced the ionized [Ca2+]. Actual concentration of left over calcium was calculated using EGTA - Free Calcium concentration calculator - a web based program by entering the experimental constants furnished in the literature source Mahaney et al Biophysical Journal 78(3) 1306-1323. Calculator program can be found at:- http://www.stanford.edu/~cpatton/webmaxclitel15.htm ************************************************************ |
| 3 | Ca2E1_prime | 0 | 0.0016667 | No |
| | 2 Ca bond state of the Enzyme SERCA2a. |
| 4 | CaE1 | 0 | 0.0016667 | No |
| | 1 Ca bond state of SERCA2a |
| 5 | CaE1_prime | 0 | 0.0016667 | No |
| | Intermediate form which is result of conformational changes in the enzymes which makes it to bind to 2nd Ca in the next step |
| 6 | CaE1_prime_ATP | 0 | 0.0016667 | No |
| | Phosporylated form of the Ca-SERCA2a. |
| 7 | E1 | 0 | 0.0016667 | No |
| | Intermediate from of Ca-ATPase (SERCA2a) which is ready to bind to first Ca. |
| 8 | E2 | 0.1509 | 0.0016667 | No |
| | Concentration was calculatd from the literature source
Mahaney et al. ( 2000 Mar) Biophys J. 78(3) 1306-23.
In case of EP formation and Pi liberation E2 is set to calculated concentration (0.1509uM), in case EP decompostion E2 is set to 0uM.
Calculations of initial SERCA2a concentration:
Data given: 8.3% of the total protein is SERCA2a. 0.2mg of SF21 microsome was taken. Molecular wt of SERCA2a is 110,000 Da. Thus E2 conc in moles is (8.3 * 0.2 * 1e-6 ) / 11 = 0.1509uM. |
| 9 | EP | 0 | 0.0016667 | No |
| | Formation and decomposition of phosphoenzyme is monitured on a time course under 3 condition.
1. SERCA2a alone.
2. SERCA2a + Wild type phospholamban.
3. SERCA2a + L37A phospholamban. |
| 10 | EPi | 0 | 0.0016667 | No |
| | Phosphoenzyme decompostion intermeditate state. |
| 11 | MgATP | 1 | 0.0016667 | No |
| | Conc of ATP was taken from the literature source Mahaney et al. ( 2000 Mar) Biophys J. 78(3) 1306-23. |
| 12 | pi | 0 | 0.0016667 | No |
| | Phosphate liberated due to EP decompostion and is propostion to EP steady state level. |
