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 comp lex and inhibit the polymerization to F-actin under high salt conditio ns. The interactions between actin and beta-thymosins have been studie d under polymerization conditions using actin labeled by a fluorescent reporter group at Cys374. Instead of labeling actin we employed equil ibrium centrifugation of unlabeled G-actin, viscometry, and chemical c ross-linking to investigate the interactions with several beta-thymosi ns, oxidized thymosin beta(4) and N-terminally truncated beta(4). The apparent dissociation constants for actin from bovine heart and beta-t hymosins were 2.5, 0.1, and 2.7 mu M for thymosin beta(4), [Ala1]beta( 4)(beta(4)(Ala)), and beta(10), respectively. Comparable apparent diss ociation constants were obtained for the interaction of G-actin from r abbit skeletal muscle and thymosin beta(4) or beta(4)(Ala). In rabbits thymosin beta(4)(Ala) replaces beta(4) being different in amino acid residue I only. The apparent dissociation constant of thymosin beta(10 ), with actin from rabbit skeletal muscle, however, is about 10% of th e value obtained with actin from bovine heart. Oxidation of thymosin b eta(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-termin us increase apparent dissociation constants to 38-53 mu M. Truncation of the first 23 amino acid residues [beta(4)-(24-43)] abolishes intera ction with G-actin completely. Therefore, amino acid residues between position 13 and 14 are necessary for 1-ethyl-3[3-(dimethyl-aminopropyl )-carbo cross-linking of G-actin. In spite of comparable apparent diss ociation constants between actin and thymosin beta(4)-sulfoxide or bet a(4)-(7-43) or beta(4)-(13 -43), only beta(4)-sulfoxide and not the tr uncated 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 polymer ization as analyzed by viscometry. While the apparent dissociation con stant of the actin/thymosin beta(4) complex generated from a preformed actin/DNase-I complex is 160 mu M, a fivefold excess of DNase I over the preformed actin/thymosin-beta(4) complex is necessary to observe a comparable dissociation constant.