, Volume 7, Issue 1, pp 33–36 | Cite as

Insulin-like growth factor binding protein (IGFBP) substrate zymography

A new tool to identify and characterize IGFBP-degrading proteinases
  • John L. Fowlkes
  • Kathryn M. Thrailkill
  • Delila M. Serra
  • Hideaki Nagase
Original Articles


Insulin-like growth factor binding protein (IGFBP) degrading proteinase activities have been described in biological fluids and conditioned media from numerous cell lines. To identify and characterize IGFBP-degrading proteinases, our laboratory has developed IGFBP substrate zymography. Herein, we illustrate how IGFBP substrate zymography can be used both to identify candidate IGFBP-degrading proteinases and characterize their degradative capabilities. For this purpose, human matrix metalloproteinase-3 (MMP-3), a proteinase that degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through a polyacrylamide gel containing IGFBP-3 as substrate and then analyzed for its ability to degrade the substrate into immunoreactive fragments that were abssorbed onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zymography was capable of detections as little as 20 ng of human MMP-3, demonstrating a sensitivity similar to casein substrate zymography. Using the zymogram as a template, MMP-3 was identified in a standard SDS-polyacrylamide gel run in parallel with the zymogram, and the corresponding area of the gel was excised. Electroelution of the gel slice yielded active MMP-3 when examined by casein substrate zymography. Furthermore, digestion of IGFBP-3 in solution by the electroeluted MMP-3 revealed the same fragmentation pattern of the binding protein as that produced by MMP-3, which had not been electroeluted. Together, these studies demonstrate that IGFBP substrate zymography can be a useful tool for both the identification and the characterization of IGFBP-degrading proteinases.

Key Words

Insulin-like growth factor binding proteins zymography proteinase matrix metalloproteinase 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rechler, M. M. (1993).Vitamin. Horm. 47, 1–114.CrossRefGoogle Scholar
  2. 2.
    Bach, L. A. and Rechler, M. M. (1995).Diabetes Rev. 3, 38–61.Google Scholar
  3. 3.
    Jones, J. I. and Clemmons, D. R. (1995).Endocr. Rev.,16, 3–34.PubMedCrossRefGoogle Scholar
  4. 4.
    Rechler, M. M. (1995). In:Molecular Endocrinology: Basic Concepts and Clinical Correlations. Weintraub, B. D. (ed.) Raven: New York, pp. 155–180.Google Scholar
  5. 5.
    Thrailkill, K. M., Quarles, L. D., Nagase, H., Suzuki, K., Serra, D. M., and Fowlkes, J. L. (1995).Endocrinology 136, 3527–3533.PubMedCrossRefGoogle Scholar
  6. 6.
    Fowlkes, J. L., Enghild, J. J., Suzuki, K., and Nagase, H. (1994).J. Biol. Chem.,269, 25,742–25,746.Google Scholar
  7. 7.
    Fowlkes, J. L., Suzuki, K., Nagase, H., and Thrailkill, K. (1994).Endocrinology 135, 2810–2813.PubMedCrossRefGoogle Scholar
  8. 8.
    Cohen, P., Graves, C. B. G., Peehl, D. M., Kamarei, M., Giudice, L. C., and Rosenfeld, R. G. (1992).J. Clin. Endocrinol. Metab. 75, 1046–1053.PubMedCrossRefGoogle Scholar
  9. 9.
    Campbell, P. G., Novak, J. F., Yanosick, T. B., and McMaster, J. H. (1992).Endocrinology 130, 1401–1412.PubMedCrossRefGoogle Scholar
  10. 10.
    Conover, C. A. and DeLeon, D. D. (1994).J. Biol. Chem. 269, 7076–7080.PubMedGoogle Scholar
  11. 11.
    Fowlkes, J. L. (1994).Endocrinology J. 2, 63–68.Google Scholar
  12. 12.
    Sommer, A., Maack, C. A., Spratt, S. K., Mascarenhas, D., Tressel, T. J., Rhodes, E. T., Lee, R., Roumas, M., Tatsuro, G. P., Flynn, J. A., Gerber, N., Taylor, J., Cudney, H., Nanney, L., Hunt, T. K., and Spencer, E. M. (1991). In:Modern Concepts of Insulin-Like Growth Factors. Spencer, E. M. (ed.) Elsevier: New York, pp. 715–728.Google Scholar
  13. 13.
    Nagase, H., Suzuki, K., Cawston, T. E., and Brew, K. (1997).Biochem. J. 325, 163–167.PubMedGoogle Scholar
  14. 14.
    Laemmli, U. K. (1970)Nature 227, 680–685.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1997

Authors and Affiliations

  • John L. Fowlkes
    • 1
  • Kathryn M. Thrailkill
    • 1
  • Delila M. Serra
    • 2
  • Hideaki Nagase
    • 3
  1. 1.Department of PediatricsUniversity of Kentucky College of MedicineLexington
  2. 2.Department of PediatricsDuke University Medical CenterDurham
  3. 3.Department of Biochemistry and Molecular BiologyUniversity of Kansas Medical CenterKansas City

Personalised recommendations