Related Experiment Videos
Epidermal growth factor binding to human alpha 2-macroglobulin. Implications for alpha 2-macroglobulin-growth factor
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Biochemistry
|August 10, 1993
Summary
Epidermal growth factor (EGF) covalently binds to activated alpha-2-macroglobulin (alpha 2M) via its amino group, with binding reduced at higher pH. This interaction is dependent on proteinase activation of alpha 2M.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Alpha-2-macroglobulin (alpha 2M) is a large plasma proteinase inhibitor.
- Epidermal growth factor (EGF) is a key regulator of cell growth and differentiation.
- The interaction between alpha 2M and EGF has implications for understanding proteinase regulation and growth factor signaling.
Purpose of the Study:
- To investigate the binding mechanism of epidermal growth factors (EGF) to human alpha-2-macroglobulin (alpha 2M).
- To determine the nature of the covalent linkage between EGF and alpha 2M.
- To explore the influence of pH and proteinase activation on this interaction.
Main Methods:
- Binding studies using 125I-labeled human and mouse EGF with human alpha 2M.
- Analysis of binding under various conditions: presence of proteinase (human neutrophil elastase), methylamine activation, and varying pH.
- Chemical modification of EGF (reductive methylation) to assess the role of the alpha-amino group.
- Dithiothreitol reduction to distinguish between reducible and nonreducible covalent bonds.
Main Results:
- EGF binding to alpha 2M was significantly enhanced in the presence of proteinase, forming predominantly nonreducible covalent bonds.
- Methylamine activation of alpha 2M resulted in greatly reduced EGF binding compared to proteinase activation.
- Reductive methylation of EGF's alpha-amino group largely abolished covalent binding, indicating its crucial role.
- Binding affinity decreased with increasing pH, primarily affecting the covalent nonreducible component.
Conclusions:
- EGF reacts with the thiol ester of proteinase-activated alpha 2M.
- The primary binding mechanism involves nucleophilic attack by the EGF alpha-amino group.
- A secondary mechanism may involve sulfide-disulfide exchange.
- The observed pH dependence suggests competition between EGF and hydroxide for the reactive thiol ester site.