C-terminal truncation of thymosin beta(10) by an intracellular protease and its influence on the interaction with G-actin studied by ultrafiltration

Two beta-thymosins are expressed in most mammalian tissues. We detected small amounts of a third peptide in extracts of rabbit spleen, The portion of this peptide increased when the tissue was first frozen and then thawed at 4 degrees C, Small amounts of the peptide are also present in cells from suspension cultures homogenized immediately in diluted perchloric acid, By means of amino acid analysis and MALDI-mass spectroscopy this peptide was identified to be a C-terminally truncated form of thymosin beta(10). Having studied the formation in more detail we found that after a 4-h thaw at 4 degrees C all thymosin beta(10) was truncated to thymosin beta(10)(1-41), which was further degraded during the next 20 h. On the other hand, thymosin beta(4)(Ala), the second beta-thymosin being present in rabbit spleen, was not truncated or degraded even after 22 h. It might be possible that in vivo a truncated form of thymosin beta(10) is formed by a carboxydipeptidase while thymosin beta(4)(Ala) is rather stable against proteolytic modification, By using a newly designed ultrafiltration assay, we determined the dissociation constants of the complexes of G-actin and these three beta-thymosins to be 0.28, 0.72, and 0.94 mu M for thymosin beta(4)(Ala), beta(10), and thymosin beta(10)(1-41), respectively. The complex with beta(4)(Ala) is unambiguously more stable than the complex with beta(10) or beta(4) (0.81 mu M). The change in the dissociation constant generated by the truncation of the two C-terminal amino acid residues of beta(10) is small but statistically significant, This demonstrates that even the very last amino acid residues at the C-terminus of beta-thymosins are involved in the interaction with G-actin. (C) 1997 Federation of European Biochemical Societies.