MAPPING THE BINDING-SITE OF THYMOSIN BETA(4) ON ACTIN BY COMPETITION WITH G-ACTIN BINDING-PROTEINS INDICATES NEGATIVE COOPERATIVITY BETWEENBINDING-SITES LOCATED ON OPPOSITE SUBDOMAINS OF ACTIN

The beta-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering fa ctors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin beta(4) was determined by analysing the binding of thymosin beta(4) to actin complexed with DNase I, gelsolin or gelsoli n segment 1. Binding was analysed by determining the increase in the c ritical concentration of actin polymerization by native gel electropho resis or chemical cross-linking. The formation of a ternary complex in cluding thymosin beta(4) should indicate that the actin-binding protei ns attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negativ e co-operative linkage between the two binding sites. Competition of t hymosin beta(4) for actin binding was observed in the presence of inta ct gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin beta(4) binds to a site close to or identical with the gelsolin segment 1-binding-site. The ternary complex of actin-DNase I- thymosin beta(4) was obtained only when using the chemically cross-lin ked actin-thymosin beta(4) complex, indicating that thymosin beta(4) i s dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin beta(4) by DNase I binding to actin is c aused by negative co-operativity between their spatially separated bin ding sites on actin. A similar negative co-operativity was observed be tween DNase I and gelsolin segment 1 binding to actin. The results the refore indicate that the respective binding sites for DNase I and segm ent 1 on subdomains 1 and 2 of actin are linked in a negative co-opera tive manner.