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 detecte d small amounts of a third peptide in extracts of rabbit spleen, The p ortion of this peptide increased when the tissue was first frozen and then thawed at 4 degrees C, Small amounts of the peptide are also pres ent in cells from suspension cultures homogenized immediately in dilut ed perchloric acid, By means of amino acid analysis and MALDI-mass spe ctroscopy this peptide was identified to be a C-terminally truncated f orm 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) wa s truncated to thymosin beta(10)(1-41), which was further degraded dur ing the next 20 h. On the other hand, thymosin beta(4)(Ala), the secon d beta-thymosin being present in rabbit spleen, was not truncated or d egraded even after 22 h. It might be possible that in vivo a truncated form of thymosin beta(10) is formed by a carboxydipeptidase while thy mosin beta(4)(Ala) is rather stable against proteolytic modification, By using a newly designed ultrafiltration assay, we determined the dis sociation constants of the complexes of G-actin and these three beta-t hymosins to be 0.28, 0.72, and 0.94 mu M for thymosin beta(4)(Ala), be ta(10), and thymosin beta(10)(1-41), respectively. The complex with be ta(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 gener ated by the truncation of the two C-terminal amino acid residues of be ta(10) is small but statistically significant, This demonstrates that even the very last amino acid residues at the C-terminus of beta-thymo sins are involved in the interaction with G-actin. (C) 1997 Federation of European Biochemical Societies.