Role of sequence 18-29 on actin in actomyosin interactions.

Affinity-purified polyclonal antibodies prepared against a synthetic peptide corresponding to sequence 18-29 from the N-terminus of rabbit alpha-skeletal actin reacted with G- and F-actin. Epitope mapping experiments with thrombin and hydroxylamine cleaved actin, and immunochemical assays verified the specificity of antibodies for the 18-29 sequence on actin. The binding of up to 0.5 mol of IgG per mole of actin did not affect the rigor binding of myosin subfragment 1 (S-1) to actin. Similarly, the binding of IgG to actin was not changed by a complete saturation of actin by S-1. In contrast to this, the weak acto-S-1 interactions in the presence of ATP were strongly inhibited by the 18-29 antibodies. At 25 degrees C, the acto-S-1 ATPase activity was inhibited by IgG stronger than the binding of S-1.ATP gamma S to actin. Thus, at this temperature, a catalytic inhibition of the acto-S-1 system appears to account at least in part for the antibody effect. Acto-S-1 ATPase activities at 25 degrees C were inhibited also by F(ab)(18-29). At 5 degrees C, the acto-S-1 ATPase activity and the binding of S-1.ATP to actin were inhibited approximately to the same extent by IgG(18-29). These results are discussed in terms of S-1 binding sites on actin and the possible role of sequence 18-29 in actomyosin interactions.