Abstract:
:We have examined the binding of 125I-labeled human and mouse epidermal growth factors (EGF) to human alpha 2-macroglobulin (alpha 2M). In the presence of human neutrophil elastase, both mouse and human EGF bound to alpha 2M, whereas little binding was found to native alpha 2M. Binding was found to be predominantly covalent and mostly nonreducible by dithiothreitol. Greatly reduced binding was found when methylamine rather than proteinase was used to convert native alpha 2M to fast-form alpha 2M. Pretreatment of native alpha 2M with either proteinase or methylamine greatly reduced binding of EGF. Titration of human 125I-EGF into native alpha 2M, in the presence of 2 equiv of proteinase, gave a gradual increase in EGF binding as a function of EGF concentration. Between 0.8 and 1.0 equiv of hEGF were bound per alpha 2M tetramer when 30 equiv of EGF were used. Reductive methylation of the alpha-amino group of mouse EGF eliminated most of the non-disulfide-mediated covalent binding. The pH dependence of binding of both mouse and human EGF to alpha 2M was examined and showed more EGF bound at pH 6 than at pH 9. The reduction in binding with increasing pH was mostly for the covalent nonreducible component. These results suggest that EGF can react with the reactive thiol ester of proteinase-activated alpha 2M by nucleophilic attack of the alpha-amino group and to a lesser extent by sulfide-disulfide exchange with the free SH of the cleaved thiol ester. The pH dependence is thought to result from competition with hydroxide for thiol ester cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Gettins PG,Crews BCdoi
10.1021/bi00082a012subject
Has Abstractpub_date
1993-08-10 00:00:00pages
7916-21issue
31eissn
0006-2960issn
1520-4995journal_volume
32pub_type
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