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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

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 beta 4 (T beta 4), 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 KD value of the thymosin beta 4 complex as 0.8 microM, a value that is in agreement with previous determinations. In addition to the main compound thymosin beta 4, bovine tissue contains a related peptide, thymosin beta 9 (T beta 9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T beta 9, similar to T beta 4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a KD value of 1.1 microM the affinity of T beta 9 is in the same range as that of T beta 4, suggesting that T beta 9, like T beta 4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of T beta 9 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 T beta 9 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 T beta 9 must be located close to the carboxy-terminus.[1]

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