Figure 7.

Equilibrium ⁴⁵Ca²⁺ binding measurements. ⁴⁵Ca²⁺ binding to deoxycholate-purified native sarcoplasmic reticulum vesicles and to DOPC-reconstituted yeast-expressed E309Q was measured by filtration. Filters, deposited with the protein samples, were perfused with 20 ml medium containing 100 mM MOPS/Tris (pH 7.2), 100 mM KCl, 1 mM MgCl₂, 55 μM CaCl₂ with radiolabelled ⁴⁵Ca²⁺, and various concentrations of EGTA, giving the free ⁴⁵Ca²⁺ concentrations indicated on the abscissa. Circles, wild type; triangles, E309Q. The lines show the best fits of the Hill equation, Y=Y_max · [Ca²⁺]^h/(K_0.5^h+[Ca²⁺]^h), to the data giving the following affinity constants (K_0.5) and Hill coefficients (h): wild type, K_0.5=0.82±0.16 μM, h=1.38 (n=2, 28 data points); E309Q, K_0.5=2.42±1.96 μM, h=0.91 (n=2, 28 data points). In each Hill fit, the Y_max was defined by the amount of ⁴⁵Ca²⁺ bound following perfusion with EGTA-free medium (hence, with 55 μM free Ca²⁺).