C-terminal truncation of thymosin beta10 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 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.
Huff
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T
Institut für Biochemie, Medizinische Fakultät, Universität Erlangen-Nürnberg, Erlangen, Germany.
Müller
C S
CS
Hannappel
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D000199:Actins / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013154:Spleen / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013947:Thymosin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014462:Ultrafiltration
D000199:Actins; D000596:Amino Acids; D010446:Peptide Fragments; D013947:Thymosin; C039302:thymosin beta(10); D010450:Endopeptidases