FIGURE 6.

Time course of EP formation through utilization of [³²P]P_i by WT SERCA (A) and E309Q mutant (B) and decay (C and D) of [³²P]phosphoenzyme after a chase with nonradioactive P_i. Microsomes derived from COS1 cells expressing SERCA or E309Q mutant were incubated with 50 mm MES, pH 6.0, 20% Me₂SO₄, 10 mm MgCl₂, and 2 mm EGTA. The reaction was started by the addition of 50 μm [³²P]P_i, quenched at different times, and processed for determination of radioactive protein by electrophoretic analysis and detection of radioactivity (see “Experimental Procedures”). For determination of decay, the protein was incubated with 50 μm [³²P]P_i as described above at 10 °C for 2 min, and then a 20-fold dilution was obtained with a medium containing 50 mm MES, pH 6.0, 10 mm MgCl₂, 2 mm EGTA, and 1 mm nonradioactive P_i at 10 or 30 °C. Serial samples were taken at sequential times for acid quenching and determination of residual radioactive protein by electrophoretic analysis and detection of radioactivity. The experimental points are averages of values obtained in three to four different experiments.