Characterization of the binary interaction between human erythrocyte protein 4.1 and actin

The binary interaction between human erythrocyte protein 4.1 and rabbit skeletal muscle F-actin was examined by rapid pelleting of the binary complexes. The binding curves show that the reaction was saturable at approximately one protein 4.1 molecule/actin monomer. The reaction was highly co-operative, displaying a Hill coefficient close to 2. Using a fixed concentration of radiolabelled protein 4.1, and varying the concentration of F-actin, the apparent molar association constant, Ka, was observed to range from 5 x 10(4) M-1 to > 10(6) M-1. The binary interaction between erythrocyte spectrin and actin was also observed to be co-operative under the same conditions. The rate of reaction between protein 4.1 and actin was temperature sensitive in a manner consistent with a high energy of activation. The pelleting assay also showed that the concentration of actin was reduced in the supernatant in the presence of protein 4.1 compared with actin alone, indicating that the critical concentration of actin was lowered in the presence of protein 4.1. Polyvalent anions disrupted the binary interaction between F-actin and protein 4.1, the disruption being consistent with the number of negative charges on these anions at pH 7.5. We postulate that the co-operativity of the binding of protein 4.1 to actin results from a protein 4.1 molecule binding to a single monomer within the filament structure which then promotes conformational changes allowing further protein 4.1 binding. The demonstration of a specific binary association between protein 4.1 and actin suggests that this interaction contributes significantly to the stabilization of the spectrin-actin-protein-4.1 ternary complex.