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Related Experiment Videos

Platelet-derived growth factor. I. High yield purification and evidence for multiple forms

E W Raines, R Ross

    The Journal of Biological Chemistry
    |May 10, 1982
    PubMed
    Summary

    Researchers purified platelet-derived growth factor (PDGF) from human platelets, finding it stimulates cell DNA synthesis. The active PDGF molecule comprises two polypeptide chains, and reducing disulfide bonds inactivates its growth-promoting activity.

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    Area of Science:

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Background:

    • Platelet-derived growth factor (PDGF) is a potent mitogen involved in cell growth and proliferation.
    • Understanding PDGF's structure is crucial for elucidating its biological functions and therapeutic potential.

    Purpose of the Study:

    • To purify PDGF from human platelet-rich plasma.
    • To characterize the molecular weight and subunit composition of active PDGF.
    • To investigate the role of disulfide bonds in PDGF's mitogenic activity.

    Main Methods:

    • Five-step purification of PDGF from human platelet-rich plasma.
    • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions.
    • Chemical cleavage of disulfide bonds (S-sulfonation, dithiothreitol, performic acid oxidation).

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  • Two-dimensional 125I-peptide mapping.

    • Main Results:

    • PDGF was purified with a 21% overall yield, achieving a 500,000-fold purification over serum.
    • Purified PDGF stimulated DNA synthesis in Swiss 3T3 cells.
    • Nonreduced PDGF exhibited four molecular species (31,000, 29,000, 28,500, 27,000 Da).
    • Reduction of disulfide bonds yielded three inactive chains (17,500, 16,000, 14,400 Da) and abolished mitogenic activity.
    • Peptide mapping suggested PDGF consists of two polypeptide chains: a 14,400 Da chain and either a 17,500 or 16,000 Da chain.

    Conclusions:

    • PDGF is a potent stimulator of DNA synthesis.
    • The mitogenic activity of PDGF is dependent on its native disulfide-bonded structure.
    • PDGF is proposed to be a heterodimer composed of two distinct polypeptide chains.