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Method to analyze collagenase and gelatinase activity by fibroblasts in culture

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Summary

The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments.

A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)NH2, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.

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References

  1. Aimes, R. T.; Quigley, J. P. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J. Biol. Chem. 270:5872–5876; 1995.

    Article  PubMed  CAS  Google Scholar 

  2. Becherer, J. D.; Howe, A.; Patel, I.; Wisely, B.; LeVine, H.; McGeehan, G. M. Characterization of the collagen and TIMP binding domains in collagenase using truncated enzymes and a chimeric matrix metalloproteinase. J. Cell Biochem. Suppl. 15G:139; 1991.

    Google Scholar 

  3. Berman, J.; Green, M.; Sugg, E.; Anderegg, R.; Millington, D. S.; Norwood, D. L.; McGeehan, J.; Wiseman, J. Rapid optimization of enzyme substrates using defined substrate mixtures. J. Biol. Chem. 267:1434–1437; 1992.

    PubMed  CAS  Google Scholar 

  4. Bickett, D. M.; Green, M. D.; Berman, J.; Dezube, M.; Howe, A.; Brown, P. J.; Roth, J. T.; McGeehan, G. M. A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). Anal. Biochem. 212:58–64; 1993.

    Article  PubMed  CAS  Google Scholar 

  5. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254; 1976.

    Article  PubMed  CAS  Google Scholar 

  6. Cawston, T. E.; Barrett, A. J. A rapid and reproducible assay for collagenase using I-14C acetylated collagen. Anal. Biochem. 99:340–345; 1979.

    Article  PubMed  CAS  Google Scholar 

  7. Dean, D. D.; Woessner, J. F. A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]-acetylated collagen. Anal. Biochem. 148:174–181; 1985.

    Article  PubMed  CAS  Google Scholar 

  8. Frieje, J. M.; Diez-Itza, I.; Balbin, M.; Sanchez, L. M.; Blasco, R.; Tolivia, J.; Lopez-Otin, C. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J. Biol. Chem. 269:16766–16773; 1994.

    Google Scholar 

  9. Heussen, C.; Dowdle, E. B. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102:196–202; 1980.

    Article  PubMed  CAS  Google Scholar 

  10. Jeffrey, J. J. Collagen degradation. In: Cohen, I. K.; Diegelman, R. F.; Lindblad, W. J., ed. Wound healing: biochemical and clinical aspects. Philadelphia: W. B. Saunders Co.; 1992:177–194.

    Google Scholar 

  11. Kleiner, D. E.; Stetler-Stevenson, W. G. Structural biochemistry and activation of matrix metalloproteinases. Curr. Opin. Cell Biol. 5:891–897; 1993.

    Article  PubMed  CAS  Google Scholar 

  12. Knight, C. G.; Willenbrock, F.; Murphy, G. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett. 296:263–266; 1992.

    Article  PubMed  CAS  Google Scholar 

  13. Nagai, Y.; Lapiere, C. M.; Gross, J. Tadpole collagenase. Preparation and purification. Biochemistry 5:3123–3130; 1966.

    Article  PubMed  CAS  Google Scholar 

  14. Stack, M. S.; Gray, R. D. Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide. J. Biol. Chem. 264:4277–4281; 1989.

    PubMed  CAS  Google Scholar 

  15. Weingarten, H.; Martin, R.; Feder, J. Synthetic substrates of vertebrate collagenase. Biochemistry 24:6730–6734; 1985.

    Article  PubMed  CAS  Google Scholar 

  16. Yager, D. R.; Zhang, L. Y.; Liang, H. X.; Diegelmann, R. F.; Cohen, I. K. Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids. J. Invest. Dermatol. 107:743–748; 1996.

    Article  PubMed  CAS  Google Scholar 

  17. Yaron, A.; Carmel, A.; Katchalski-Katzir, E. Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Anal. Biochem. 95:228–235; 1979.

    Article  PubMed  CAS  Google Scholar 

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Author notes

  1. Lisa J. Gould

    Present address: Medical College of Wisconsin, Plastic and Reconstructive Surgery, 9200 West Wisconsin Avenue, 53226, Milwaukee, Wisconsin

Authors and Affiliations

  1. Laboratory of Tissue Repair, Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical College of Virginia Campus of Virginia Commonwealth University, 23298-0117, Richmond, Virginia

    Lisa J. Gould, Dorne R. Yager & Robert F. Diegelmann Ph.D.

  2. Biochemistry Department, Glaxo Inc. Research Institute, 27709, Research Triangle, North Carolina

    Gerard M. McGeehan

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  1. Lisa J. Gould
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  2. Dorne R. Yager
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  3. Gerard M. McGeehan
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  4. Robert F. Diegelmann Ph.D.
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Gould, L.J., Yager, D.R., McGeehan, G.M. et al. Method to analyze collagenase and gelatinase activity by fibroblasts in culture. In Vitro Cell.Dev.Biol.-Animal 35, 75–79 (1999). https://doi.org/10.1007/s11626-999-0004-x

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  • DOI: https://doi.org/10.1007/s11626-999-0004-x

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