FIGURE 2.

Kinetics of the dissociation of Ca²⁺ at the cytoplasmically facing sites. Quench-flow experiments were carried out by the mixing protocol illustrated by the diagram above the panels using a QFM-5 module at 25 °C. The microsomal enzyme, preincubated in 40 mm MES/Tris (pH 6.0), 80 mm KCl, 5 mm MgCl₂, and 100 μm CaCl₂ (to accumulate Ca₂E1), was mixed with an equal volume of 40 mm MES/Tris (pH 6.0), 80 mm KCl, 5 mm MgCl₂, and 4 mm EGTA, to initiate Ca²⁺ dissociation. At the indicated time intervals the amount of phosphorylatable Ca₂E1 remaining was determined by adding the double volume of 40 mm MES/Tris (pH 6.0), 80 mm KCl, 5 mm MgCl₂, 2 mm EGTA, and 10 μm [γ-³²P]ATP, followed by acid quenching 34 ms later. To obtain the point corresponding to zero time (in each case taken as 100%), 4 mm EGTA was replaced by 100 μm CaCl₂. All data points are shown. The lines show the best nonlinear regression fits of a monoexponential decay function, giving the rate constants listed in Table 2. The broken lines represent the curves corresponding to wild type SERCA1a and SERCA2b from the upper left panel.