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 thymosinp4 (T/94), 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 Kn value of the thymosin @l complex as 0.8 PM, a value that is in agreement with previous determinations. In addition to the main compound thymosin/?4, bovine tissue contains a related peptide, thymosin p (Tp9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T/$?, similar to Tp4, forms a 1:l complex with monomeric actin, and hereby inhibits actin polymerization. With a K, value of 1.1 FM the affinity of TjT9 is in the same range as that of Tp4, suggesting that T@9, like T/?4, contributes to maintaining the pool of monomeric. actin in bovine non-muscle cells. Further proof of the interaction of T/I9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslink~g experiments. Using different crosslinking reagents, like water-soluble ~~iimide (EDC), ~-maleimido~~oyl-~-hydroxys~nimi~te (MBS), and disu~inimidylsu~rate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tfi9 could be identified by imm~oblot~ng. 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/39 must be located close to the carboxy-terminus.

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β-Thymosins, a group of highly homologous peptides consisting of about 40 amino acid residues, were found to be distributed from mammals up to echinoderms. Althogh they have first been isolated from mammalian thymus tissue preparations, their occurrance is not organ-specific and they are present even in different types of cells. For thymosinβ 4 several biological activities have been reported, stating that this peptide acts as a thymus peptide hormone and is also involved in the neuroendocrine and immune system. However, it was recently demonstrated that thymosinβ 4 has actin-sequestering properties and therefore might play an important role in the regulation of the microfilament system. This fact gives a new outlook on the real biological function ofβ-thymosins.

Thymosin 84 forms a 1: 1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin-thymosin /I4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin Sl were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin 84 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non-muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin-stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.