Use of bimanyl actin derivative (TMB-actin) for studying complexation of β-thymosins: Inhibition of actin polymerization by thymosin β9

• Published in: FEBS letters 1993-08, Vol.329 (1), p.9-12
• Main Authors: Heintz, Daniela, Reichert, Andreas, Mihelic, Mirna, Voelter, Wolfgang, Faulstich, Heinz
• Format: Article
• Language: English
• Subjects: Bimanylfluorescence; Binding constant; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Interactions. Associations; Intermolecular phenomena; Molecular biophysics; Muscle actin; Polymerization inhibition; Thiol spcificity; β-Thymosin
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80181-S

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 β4 (Tβ4), 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 β4 complex as 0.8 μM, a value that is in agreement with previous determinations. In addition to the main compound thymosin β4, bovine tissue contains a related peptide, thymosin β9 (Tβ9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that Tβ9, similar to Tβ4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a K d value of 1.1 μM the affinity of Tβ9 is in the same range as that of Tβ4, suggesting that Tβ9, like Tβ4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of Tβ9 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 Tβ9 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 Tβ9 must be located close to the carboxy-tenninus.