USE OF BIMANYL ACTIN DERIVATIVE (TMB-ACTIN) FOR STUDYING COMPLEXATION OF BETA-THYMOSINS - INHIBITION OF ACTIN POLYMERIZATION BY THYMOSIN-BETA-9

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosin beta4 (Tbeta4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the K(D) value of the thymosin beta4 complex as 0.8 muM, a value that is in agreement with previous determinations. In addition to the main compound thymosin beta4, bovine tissue contains a related peptide, thymosin beta9 (Tbeta9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that Tbeta9, similar to Tbeta4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a K(D) value of 1.1 muM the affinity of Tbeta9 is in the same range as that of Tbeta4, suggesting that Tbeta9, like Tbeta4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of Tbeta9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water-soluble carbodiimide (EDC), m-maleimidobenzoyl-N-hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tbeta9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin. the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of Tbeta9 must be located close to the carboxy-terminus.