Interactions of beta-thymosins, thymosin beta 4-sulfoxide, and N-terminally truncated thymosin beta 4 with actin studied by equilibrium centrifugation, chemical cross-linking and viscometry.

All beta-thymosins studied interact with G-actin in a bimolecular complex and inhibit the polymerization to F-actin under high salt conditions. The interactions between actin and beta-thymosins have been studied under polymerization conditions using actin labeled by a fluorescent reporter group at Cys374. Instead of labeling actin we employed equilibrium centrifugation of unlabeled G-actin, viscometry, and chemical cross-linking to investigate the interactions with several beta-thymosins, oxidized thymosin beta 4 and N-terminally truncated beta 4. The apparent dissociation constants for actin from bovine heart and beta-thymosins were 2.5, 0.1, and 2.7 microM for thymosin beta 4, [Ala1]beta 4(beta Ala4), and beta 10, respectively. Comparable apparent dissociation constants were obtained for the interaction of G-actin from rabbit skeletal muscle and thymosin beta 4 or beta Ala4. In rabbits thymosin beta Ala4 replaces beta 4 being different in amino acid residue 1 only. The apparent dissociation constant of thymosin beta 10 with actin from rabbit skeletal muscle, however, is about 10% of the value obtained with actin from bovine heart. Oxidation of thymosin beta 4 at Met6 (beta 4-sulfoxide) as well as truncation of 6 [beta 4-(7-43)] or 12 [beta 4-(13-43)] amino acid residues from the N-terminus increase apparent dissociation constants to 38-53 microM. Truncation of the first 23 amino acid residues [beta 4-(24-43)] abolishes interaction with G-actin completely. Therefore, amino acid residues between position 13 and 24 are necessary for 1-ethyl-3[3-(dimethyl-aminopropyl)-carbodiimide cross-linking of G-actin. In spite of comparable apparent dissociation constants between actin and thymosin beta 4-sulfoxide or beta 4-(7-43) or beta 4-(13-43), only beta 4-sulfoxide and not the truncated beta-thymosins inhibits actin polymerization, however, only at a 20-fold higher concentration than beta 4. Thus the first six amino acid residues are indispensable to inhibit salt-induced actin polymerization as analyzed by viscometry. While the apparent dissociation constant of the actin/thymosin beta 4 complex generated from a preformed actin/DNase-I complex is 160 microM, a fivefold excess of DNase I over the preformed actin/thymosin-beta 4 complex is necessary to observe a comparable dissociation constant.