Use of bimanyl actin derivative (TMB-actin) for studying complexation of beta-thymosins. Inhibition of actin polymerization by thymosin beta 9.
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 thymosin beta 4 (T beta 4), 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 KD value of the thymosin beta 4 complex as 0.8 microM, a value that is in agreement with previous determinations. In addition to the main compound thymosin beta 4, bovine tissue contains a related peptide, thymosin beta 9 (T beta 9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T beta 9, similar to T beta 4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a KD value of 1.1 microM the affinity of T beta 9 is in the same range as that of T beta 4, suggesting that T beta 9, like T beta 4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of T beta 9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water-soluble carbodiimide (EDC), m-maleimidobenzoyl-N-hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and T beta 9 could be identified by immunoblotting. 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 beta 9 must be located close to the carboxy-terminus.
Heintz
D
D
Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Reichert
A
A
Mihelic
M
M
Voelter
W
W
Faulstich
H
H
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011108:Polymers / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013388:Succinimides; D013947:Thymosin / Q000378:metabolism / Q000494:pharmacology*
D000199:Actins; D001643:Bridged Bicyclo Compounds; D003432:Cross-Linking Reagents; D011108:Polymers; D013388:Succinimides; C082720:actin, (TMB)Cys(374)-; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D013947:Thymosin; C042362:thymosin beta(9); D003850:Deoxyribonuclease I; D003545:Cysteine