FIGURE 6.

Pre-steady state behavior of SERCA (A) and CopA (B) upon sequential additions of ATP and Ca²⁺ or Cu⁺·SERCA (0.05 mg/ml) preincubated for 15 min in a medium containing 50 mm MOPS, pH 7, 80 mm KCl, 1 mm MgCl₂, 1 μm A23187 (Ca²⁺ ionophore), and 1 mm EGTA. The reaction was started at 10 °C by the addition of 25 μm [γ-³²P]ATP and, after 10 s, 1.05 mm CaCl₂ was added. CopA (0.1 mg/ml) was preincubated at 37 °C with 0.1 mm BCS for 2 h, in a reaction mixture as described under “Materials and Methods.” The reaction was started at 60 °C by the addition of 25 μm [γ-³²P]ATP and, after 10 s, 0.14 mm CuCl₂ was added. Samples were taken for determination of phosphoenzyme (•) and P_i (○) at the time intervals as indicated. Each time point shown in the graph is the average of three different determinations. Note the P_i burst in concomitance with EP reduction upon addition of copper.