Reversible binding of platelet-derived growth factor-AA, -AB, and -BB isoforms to a similar site on the "slow" and "fast" conformations of alpha 2-macroglobulin.
The mechanism by which the platelet-derived growth factor (PDGF)-binding protein, alpha 2-macroglobulin (alpha 2M), modulates PDGF bioactivity is unknown, but could involve reversible PDGF-alpha 2M binding. Herein we report that greater than 70% of 125I-PDGF-BB or -AB complexed to alpha 2M was dissociated by SDS-denaturation followed by SDS-polyacrylamide gel electrophoresis, i.e. most of the binding was noncovalent. Reduction of the PDGF.alpha 2M complex following denaturation dissociated the cytokine from alpha 2M by greater than 90%, suggesting covalent disulfide bond formation. Approximately 50% of the growth factor was dissociated by lowering the pH from 7.5 to 4.0. 125I-PDGF-BB bound alpha 2M in a time-dependent manner (t1/2 = approximately 1 h), reaching equilibrium after 4 h. The 125I-PDGF.BB/alpha 2M complex dissociated more slowly (t1/2 = approximately 2.5 h). "Slow" and "fast" alpha 2M bound nearly equal amounts of PDGF-AB or -BB. Trypsin treatment converted PDGF-BB/alpha 2M complex to the fast conformation but did not release bound 125I-PDGF-BB. All PDGF-isoforms (AA, -AB, and -BB) competed for binding with 125I-PDGF-BB binding to slow alpha 2M and fast alpha 2M-methylamine by 65-80%. Other cytokines that bind alpha 2M (transforming growth factor-beta 1 and -beta 2, tumor necrosis factor-alpha, basic fibroblast growth factor, interleukin -1 beta, and -6) did not compete for 125I-PDGF-BB binding slow alpha 2M, but transforming growth factor-beta 1 and basic fibroblast growth factor inhibited 125I-PDGF-BB binding alpha 2M-methylamine by 30-50%. The reversible nature of the PDGF.alpha 2M complex could allow for targeted PDGF release near mesenchymal cells which possess PDGF receptors.
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Related Subject Headings
- alpha-Macroglobulins
- Protein Denaturation
- Protein Conformation
- Protein Binding
- Platelet-Derived Growth Factor
- Methylamines
- Kinetics
- Hydrogen-Ion Concentration
- Humans
- Electrophoresis, Polyacrylamide Gel
Citation
Published In
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- alpha-Macroglobulins
- Protein Denaturation
- Protein Conformation
- Protein Binding
- Platelet-Derived Growth Factor
- Methylamines
- Kinetics
- Hydrogen-Ion Concentration
- Humans
- Electrophoresis, Polyacrylamide Gel