Minispectrin-actin-4.1R binding assessed by an F-actin pelleting assay. (A) Effect of protein 4.1R on minispectrin-actin interactions. F-actin (2.38μM as G-actin) was combined with increasing amounts of [¹²⁵I]-minispectrin, with or without protein 4.1R (0.38μM), and the bound minispectrin was measured after pelleting the F-actin. (B) Comparison of the amount of ternary complex formed by normal and mutant minispectrins. F-actin (2.38μM as G-actin) and protein 4.1 (0.38μM) were combined with increasing amounts of [¹²⁵I]-minispectrin or [¹²⁵I]-minispectrin-Δ13, and the bound minispectrin was measured after pelleting the F-actin. Residual binding of the minispectrin to F-actin in the absence of 4.1R was subtracted from each curve to give 4.1R-specific binding. Note the marked ineffectiveness of the minispectrin bearing the α-spectrin C-terminal deletion found in the sph^IJ mouse with unstable red cell membranes and severe hemolytic anemia (minispectrin-ΔC13).