The role of cysteine-949 in the binding of transforming growth factor-beta 1 and transforming growth factor-beta 2 to alpha 2-macroglobulin.
The reaction of alpha 2-macroglobulin (alpha 2M) with proteinases or methylamine causes a major conformational change in alpha 2M and cleavage of the alpha 2M thiol ester bonds. The resulting free Cys residues (Cys-949) contain the only free thiol groups in alpha 2M. In this investigation, we explored the role of Cys-949 in the binding of transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2 to alpha 2M-methylamine. Modification of preformed alpha 2M-methylamine with iodoacetamide did not change the binding affinity of alpha 2M-methylamine for TGF-beta 1 or TGF-beta 2; the apparent KD values were 82 nM and 10 nM, respectively. TGF-beta binding also remained unchanged when tested using an alpha 2M derivative prepared by simultaneous treatment of alpha 2M with methylamine and iodoacetamide. The slow thiol-disulfide exchange reaction that irreversibly stabilizes noncovalent growth factor-alpha 2M-methylamine complexes was completely inhibited by modification of Cys-949. These studies demonstrate that Cys-949 in alpha 2M is not essential for binding of TGF-beta 1 and TGF-beta 2 noncovalently; however, this residue plays a critical role in the covalent stabilization step of the reaction mechanism.
Crookston
K P
KP
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Gonias
S L
SL
D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides; D006801:Humans; D066298:In Vitro Techniques; D007460:Iodoacetamide / Q000737:chemistry; D007700:Kinetics; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D004220:Disulfides; D016212:Transforming Growth Factor beta; D000511:alpha-Macroglobulins; D003545:Cysteine; D007460:Iodoacetamide