Crookston K P, Gonias S L
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Biochem Biophys Res Commun. 1994 May 16;200(3):1578-85. doi: 10.1006/bbrc.1994.1631.
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.
α2-巨球蛋白（α2M）与蛋白酶或甲胺反应会导致α2M发生重大构象变化，并使α2M硫酯键断裂。产生的游离半胱氨酸残基（Cys-949）是α2M中仅有的游离巯基。在本研究中，我们探究了Cys-949在转化生长因子-β1（TGF-β1）和转化生长因子-β2（TGF-β2）与α2M-甲胺结合中的作用。用碘乙酰胺修饰预先形成的α2M-甲胺不会改变α2M-甲胺对TGF-β1或TGF-β2的结合亲和力；表观解离常数（KD）值分别为82 nM和10 nM。当使用通过同时用甲胺和碘乙酰胺处理α2M制备的α2M衍生物进行测试时，TGF-β的结合也保持不变。不可逆地稳定非共价生长因子-α2M-甲胺复合物的缓慢硫醇-二硫键交换反应被Cys-949的修饰完全抑制。这些研究表明，α2M中的Cys-949对于TGF-β1和TGF-β2的非共价结合并非必不可少；然而，该残基在反应机制的共价稳定步骤中起关键作用。
