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Related Experiment Videos

Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds

W C Kenney1, M Haniu, A C Herman

  • 1Amgen Inc., Amgen Center, Thousand Oaks, California 91320.

The Journal of Biological Chemistry
|April 22, 1994
PubMed
Summary

Intrachain disulfide bonds are crucial for Platelet-Derived Growth Factor (PDGF) conformation and activity. While interchain bonds enhance dimer stability, PDGF monomers can form active noncovalent dimers without them.

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Area of Science:

  • Molecular Biology
  • Protein Chemistry
  • Cell Signaling

Background:

  • Platelet-Derived Growth Factor (PDGF) is a mitogen for mesenchymal cells, existing as disulfide-bonded dimers (AA, BB, AB).
  • The dimeric structure was presumed essential for PDGF's biological activity.
  • Reduction of PDGF dimers leads to loss of activity and monomer formation, questioning the role of specific disulfide bonds.

Purpose of the Study:

  • To investigate the role of intrachain versus interchain disulfide bonds in PDGF-B activity and dimerization.
  • To determine if disulfide bonds are critical for PDGF's correct protein conformation and biological function.

Main Methods:

  • Site-directed mutagenesis of PDGF-B to alter cysteine residues involved in disulfide bonds.
  • Analysis of mitogenic activity using cell proliferation assays.

Related Experiment Videos

  • SDS-polyacrylamide gel electrophoresis to assess monomeric/dimeric state.
  • Light scattering and sedimentation equilibrium to determine molecular weight and dimerization state.
  • Disulfide bond analysis to confirm bond formation.

    • Main Results:

    • Mutants lacking all or key cysteines showed reduced dimerization and significantly less mitogenic activity.
    • A mutant (PDGF-B(C2,4S)) lacking only interchain disulfide bonds retained significant mitogenic activity, forming noncovalent dimers at physiological pH.
    • This mutant's activity was sensitive to low pH, dissociating into monomers, unlike wild-type PDGF-BB.

    Conclusions:

    • Intrachain disulfide bonds are critical for maintaining the correct conformation of PDGF monomers.
    • Properly folded PDGF monomers can form active noncovalent dimers even without interchain disulfide bonds.
    • Interchain disulfide bonds primarily enhance PDGF dimer stability rather than being essential for its existence or activity.