FIGURE 8.

Binding of protein 4.2 to the α-spectrin EF-domain is amplified by Ca²⁺ but not by Mg²⁺. A, shown is binding of ³⁵S-labeled protein 4.2 to GST-α18–21EF in the absence or presence of free Ca²⁺ (1.5 mm). B, shown is binding of the ¹²⁵I-labeled G₃ peptide of protein 4.2 to GST-α18–21EF (0.75 μm) in the absence or presence of free Ca²⁺ (1.5 mm). C, shown is binding of ³⁵S-labeled protein 4.2 to GST-minispectrin (0.43 μm) in the absence or presence of free Ca²⁺ (1.5 mm). D, shown is the specificity of Ca²⁺ compared with Mg²⁺ in the binding of ³⁵S-labeled protein 4.2 to GST-α18–21EF (0.75 μm). All experiments used the HEPES binding buffer, which contains 0.5 mm EGTA (see “Experimental Procedures”) with or without added CaCl₂ or MgCl₂. Free Ca²⁺ and Mg²⁺ concentrations were calculated using the EGTA Calculator.