Huff T, Müller C S, Hannappel E
Institut für Biochemie, Medizinische Fakultät, Universität Erlangen-Nürnberg, Erlangen, Germany.
FEBS Lett. 1997 Sep 1;414(1):39-44. doi: 10.1016/s0014-5793(97)00946-0.
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 beta10. Having studied the formation in more detail we found that after a 4-h thaw at 4 degrees C all thymosin beta10 was truncated to thymosin beta10(1-41), which was further degraded during the next 20 h. On the other hand, thymosin beta4Ala, 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 beta10 is formed by a carboxydipeptidase while thymosin beta4Ala 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 microM for thymosin beta4Ala, beta10, and thymosin beta10(1-41), respectively. The complex with beta4Ala is unambiguously more stable than the complex with beta10 or beta4 (0.81 microM). The change in the dissociation constant generated by the truncation of the two C-terminal amino acid residues of beta10 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.
两种β-胸腺素在大多数哺乳动物组织中表达。我们在兔脾脏提取物中检测到少量的第三种肽。当组织先冷冻然后在4℃解冻时，这种肽的含量会增加。在稀释的高氯酸中立即匀浆的悬浮培养细胞中也存在少量这种肽。通过氨基酸分析和基质辅助激光解吸电离质谱法，鉴定该肽为胸腺素β10的C末端截短形式。在更详细地研究其形成过程后，我们发现，在4℃解冻4小时后，所有胸腺素β10都被截短为胸腺素β10(1-41)，并在接下来的20小时内进一步降解。另一方面，兔脾脏中存在的第二种β-胸腺素胸腺素β4Ala即使在22小时后也没有被截短或降解。体内胸腺素β10的截短形式可能是由羧肽酶形成的，而胸腺素β4Ala对蛋白水解修饰相当稳定。通过使用新设计的超滤测定法，我们确定G-肌动蛋白与这三种β-胸腺素复合物的解离常数分别为胸腺素β4Ala 0.28μM、β10 0.72μM和胸腺素β10(1-41) 0.94μM。与β4Ala形成的复合物明显比与β10或β4（0.81μM）形成的复合物更稳定。β10的两个C末端氨基酸残基截短产生的解离常数变化很小，但具有统计学意义。这表明即使是β-胸腺素C末端的最后几个氨基酸残基也参与了与G-肌动蛋白的相互作用。
