Figure 5.

Time course and ATP concentration dependence of phosphorylation by [γ-³²P]ATP. (A, B) The time course of phosphorylation of wild-type Ca²⁺-ATPase (circles) or mutant E309Q (triangles) expressed in COS-1 cells was studied at 0°C (open symbols) or 25°C (closed symbols) by incubation in a medium containing 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 25 mM MgCl₂, 5 μM [γ-³²P]ATP, and 0.1 mM CaCl₂ (A) or 5 mM CaCl₂ (B), followed by acid quench at varying time intervals. (C, D) Phosphorylation of the wild-type Ca²⁺-ATPase (C) or mutant E309Q (D) was carried out for varying time intervals at 25°C at various [γ-³²P]ATP concentrations. The medium contained 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 5 mM MgCl₂, 0.1 mM CaCl₂, and the following concentrations of [γ-³²P]ATP: circles, 1 μM; squares, 2 μM; triangles pointing upward, 5 μM; triangles pointing downward, 10 μM; diamonds, 20 μM. In each case, the level of phosphorylation is shown relative to the total concentration of Ca²⁺-ATPase active sites present, as determined by phosphorylation with ³²P_i. The lines show the best fits of a monoexponential function, EP=EP_max·(1−e^−kt), to the data, giving the rate constants shown in Supplementary Table S1 (for panels A and B) and Supplementary Table S2 (for panels C and D).