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. Author manuscript; available in PMC: 2013 May 14.
Published in final edited form as: Biochemistry. 2008 Dec 23;47(51):10.1021/bi801024a. doi: 10.1021/bi801024a

Fig. 5.

Fig. 5

Phosphorylation, E1P formation, and the weakly-immobilized EPR signal in SERCA1 at 2°C and 0.4 M KCl. (A) The dimer model of SERCA1 in Fig. 1 was modified to include an initial step (1) in which E2/E2 (non-phosphorylated species) is converted to E2/E1 (phosphorylated species) by a slow (4 s−1), [KCl]-dependent reaction. By adding this step, reactions 1–7 in Fig. 1 become reactions 2–8. As a simplifying approximation, the rate constant controlling the hydrolysis of E2P in E1P/E2P (k6) has been assigned a value of zero because of the very slow turnover of this intermediate at 2°C (). For EP formation (filled squares), SR vesicles (.25 mg/ml) were phosphorylated by 100 µM [γ32P]ATP in standard buffer containing 0.4 M KCl. Filled circles represent E1P formation evaluated from the ADP chase experiments conducted at 10, 116 and 223 ms in Fig. 6. Open circles represent E1P formation calculated from the level of phosphorylation at different time intervals (filled squares) and the assumption that E1P is 50% of EPTOTAL. At time zero, approximately 100 µM ATP was generated by laser flash photolysis from 1 mM caged ATP, and the resulting EPR transient representing the buildup of a rotationally restricted spin label fraction monitored over time. The modified dimer scheme was used to simulate phosphorylation (filled squares), E1P accumulation (filled and open circles) and the transient EPR signal (filled triangles) using the simulation routines in MLAB and the following set of rate constants (in s−1/s−1): k1/k−1 = 4/4; k2/k−2 = 350/0; k3/k−3 = 70/0; k4/k−4 = 500/0; k5/k−5 = 4/0; k6/k−6 = 0/0. Initial conditions (in nmol/mg): [ETOTAL] = 3.8; [E2/E2] = 1.9; [E2/E1] = 1.9. The amplitude of the time-resolved EPR signal has been adjusted so that the simulated EPR signal coincides with the simulated time course of E1P formation. (B) The trimer model in Fig. 4 was used to simulate phosphorylation (filled squares), E1P formation (filled and open circles) and the transient EPR signal (filled triangles) using the MLAB simulation routines and the following set of rate constants (in s−1/s−1): k1/k−1 = 4/4; k2/k−2 = 350/0; k3/k−3 = 70/0; k4/k−4 = 500/0; k5/k−5 = 400/0; k6/k−6 = 0/0. The amplitude of the transient EPR signal has been adjusted so that the kinetic similarities in the transient EPR signal and E1P formation are readily apparent.