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Further studies on the nature of the components in serum which inhibit mouse bone collagenase

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Abstract

Horse serum prepared free of trypsin inhibitors by passing it through a column of insolubilized trypsin (trypsin covalently bound to Sepharose 4B) no longer inhibits mouse bone collagenase activity. Of the various serum fractions tested, only two (beta-lipoprotein, which is a rich source of alpha-2-macroglobulin, and alpha-globulin) significantly inhibited mouse bone collagenase activity. Only the alpha-2-macroglobulin fraction of horse serum chromatographed on Bio-gel P-150 inhibited both mouse bone collagenase activity and trypsin activity. The alpha-1-antitrypsin fraction did not inhibit mouse bone collagenase activity, but did inhibit trypsin activity. Affinity chromatography of horse serum on Sepharose 4B to which trypsin had been covalently bound revealed inhibitory activity towards both trypsin and mouse bone collagenase in the fraction containing alpha-2-macroglobulin. The results suggest that the component in serum which inhibits mouse bone collagenase activity may be identical with the trypsin inhibitor present in the alpha-2-macroglobulin fraction. Serum and mouse bone collagenase chromatographed on the Bio-gel column did not bind together and the collagenase emerged at its own elution volume without loss of enzymatic activity. This suggests that an enzyme inhibitor complex (if it exists) is easily dissociable. Similar results to those above were obtained using mouse serum.

Résumé

Du sérum de cheval, dépourvu d'inhibiteurs de la trypsine, en le faisant passer sur une colonne de trypsine insolubilisée (de la trypsine liée de façon covalente au Sepharose 4B), n'inhibe plus l'activité de la collagénase de l'os de souris. Des diverses fractions du sérum testées, seules deux d'entre elles (une beta-lipoprotéine, riche en alpha-2-macroglobuline, et une alpha-globuline) inhibitent de façon significative l'activité de la collagénase de l'os de souris. Seule la fraction alpha-2-macroblobuline du sérum de cheval, chromatographiée sur du Biogel P-150, inhibe à la fois l'activité en collagénase d'os de souris et l'activité de la trypsine. La fraction alpha-1-antitrypsine n'inhibe pas l'activité en collagénase d'os de souris, mais inhibe celle de la trypsine. La chromatographie de sérum de cheval sur Sepharose 4B, avec la trypsine qui lui est lié de façon covalente, permet de mettre en évidence une activité d'inhibition envers la trypsine et la collagénase d'os de souris dans la fraction contenant l'alpha-2-macroglobuline. Les résultats semblent indiquer que le constituant du sérum, qui inhibe l'activité de la collagénase d'os de souris, pourrait être identique avec l'inhibiteur de trypsine de la fraction alpha-2-macroglobuline. Les collagénases du sérum et de l'os de souris, chromatographiées sur la colonne de Biogel, ne se combinent pas et la collagénase apparait à son propre volume d'élution, sans perte d'activité enzymatique. Ce fait semble indiquer qu'un complexe d'inhibiteur enzymatique (s'il existe) est facilement dissociable. Des résultats similaires ont été obtenus avec du sérum de souris.

Zusammenfassung

Pferdeserum wurde von Trypsinhemmern befreit, indem es über eine Säule aus unlöslich gemachten Trypsin (d. h. Trypsin kovalent an Sepharose 4B gebunden) gegeben wurde. Dadurch verlor es seine Hemmwirkung auf die Aktivität der Mäuseknochenkollagenase. Von den verschiedenen getesteten Serum-Fraktionen hemmten nur zwei (beta-Lipoprotein, welches reich an alpha-2-Makroglobulin ist, und alpha-Globulin) die Aktivität von Mäuseknochenkollagenase signifikant. Nur die alpha-2-Makroglobulinfraktion von Pferdeserum, welche auf Biogel P-150 chromatographiert wurde, hemmte die Aktivität von Mäuseknochenkollagenase und von Trypsin. Die alpha-1-Antitrypsinfraktion hemmte die Mäuseknochenkollagenase-Aktivität nicht, dagegen die Trypsin-Aktivität. Die Affinitätschromatographie von Pferdeserum auf Sepharose 4B, an welche Trypsin kovalent gebunden worden war, zeigte eine Hemmwirkung gegen Trypsin und gegen Mäuseknochenkollagenase in der alpha-2-Makroglobulin enthaltenden Fraktion. Die Resultate lassen vermuten, daß die Komponente im Serum, welche die Mäuseknochenkollagenase-Aktivität hemmt, identisch mit dem Trypsinhemmer in der alpha-2-Makroglobulinfraktion ist. Serum und Mäuseknochenkollagenase, welche auf der Biogelsäule chromatographiert wurden, verbanden sich nicht, und die Kollagenase erschien in ihrem eigenen Elutionsvolumen und ohne Verlust von Enzymaktivität. Dies läßt vermuten, daß ein Enzymhemmkomplex (falls er existiert) leicht dissoziierbar ist. Ähnliche Resultate wurden mit Mäuseserum erhalten.

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References

  1. Bauer, E. A., Eisen, A. Z., Jeffrey, J. J.: Studies on purified rheumatoid synovial collagenasein vitro andin vivo. J. clin. Invest.50, 2056–2064 (1971).

    PubMed  Google Scholar 

  2. Bundy, H. F., Mehl, J. W.: Trypsin inhibitors of human serum. I. Standardization, mechanism or reaction, and normal values. J. clin. Invest.37, 947–955 (1958).

    PubMed  Google Scholar 

  3. Bundy, H. F., Mehl, J. W.: Trypsin inhibitors of human serum. II. Isolation of the alpha-1-inhibitor and its partial characterization. J. biol. Chem.234, 1124–1128 (1959).

    PubMed  Google Scholar 

  4. Cuatrecasas, P.: Protein purification by affinity chromatography. J. biol. Chem.245, 3059–3065 (1970).

    PubMed  Google Scholar 

  5. Dyce, B., Haverback, B. J.: Serum trypsin inhibitors in the normal and in acute pancreatitis. Amer. J. Gastroent.34, 481–486 (1960).

    PubMed  Google Scholar 

  6. Eisen, A. Z., Jeffrey, J. J., Gross, J.: Human skin collagenase. Isolation and mechanism of attack on the collagen molecule. Biochim. biophys. Acta (Amst.)151, 637–645 (1968).

    Google Scholar 

  7. Eisen, A. Z., Bloch, K. J., Sakai, T.: Inhibition of human skin collagenase by human serum. J. Lab. clin. Invest.75, 258–263 (1970).

    Google Scholar 

  8. Eisen, A. Z., Bauer, E. A., Jeffrey, J. J.: Human skin collagenase. The role of serum alpha-globulins in the control of activityin vivo andin vitro. Proc. nat. Acad. Sci. (Wash.)68, 248–251 (1971).

    Google Scholar 

  9. Evanson, J. M., Jeffrey, J. J., Krane, S. M.: Studies on collagenase from rheumatoid synovium in tissue culture. J. clin. Invest.47, 2639–2651 (1968).

    PubMed  Google Scholar 

  10. Evanson, J. M.: Collagen degradation by explanted rheumatoid synovial tissue. Chemistry and molecular biology of the intercellular matrix, vol. 3. E. A. Balazs, ed., p. 1637. New York: Academic Press 1970.

    Google Scholar 

  11. Feinstein, G.: Isolation of chicken ovoinhibitor by affinity chromatography on chymotrypsin-Sepharose. Biochim. biophys. Acta (Amst.)236, 73–77 (1971).

    Google Scholar 

  12. Fritz, H., Schult, H., Hutzel, M., Wiedlmann, M., Werle, E.: Isolierung von Protease-Inhibitoren mit Hilfe wasserunlöslicher Enzym-Harze. A. Physiol. Chem.348, 308–312 (1967).

    Google Scholar 

  13. Glimcher, M. J., Francois, C. J., Richards, L., Krane, S. M.: The presence of organic phosphorus in collagens and gelatins. Biochim. biophys. Acta (Amst.)93, 585–602 (1964).

    Google Scholar 

  14. Greene, N. D., Damian, R. T., Hubbard, W. J.: The identification of alpha-2-macroglobulin in the mouse. Biochim. biophys. Acta (Amst.)236, 659–663 (1971).

    Google Scholar 

  15. Harper, E., Bloch, K. J., Gross, J.: The zymogen of tadpole collagenase. Biochemistry10, 3035–3041 (1971).

    Article  PubMed  Google Scholar 

  16. Harris, E. D., Jr., DiBona, D. R., Krane, S. M.: Collagenases in human synovial fluid. J. clin. Invest.48, 2104–2113 (1969).

    PubMed  Google Scholar 

  17. Haverback, B. J., Dyce, B., Bundy, H. F., Wirtschafter, S. K., Edmondson, H. A.: Protein binding of pancreatic proteolytic enzymes. J. clin. Invest.41, 972–980 (1962).

    PubMed  Google Scholar 

  18. Lapiere, C. M., Gross, J.: Animal collagenase and collagen metabolism. Mechanisms of hard tissue destruction. Publication No. 75. R. Sognnaes, ed. p. 663–694, Washington, D.C.: Amer. Assoc. Advanc. Sci. 1963.

    Google Scholar 

  19. Pierce, J. A., Eisen, A. Z., Dhingra, H. K.: Relationship of antitrypsin deficiency to the pathogenesis of emphysema. Trans. Amer. Ass. Phys.82, 87–97 (1969).

    Google Scholar 

  20. Sakamoto, S., Goldhaber, P., Glimcher, M. J.: Maintenance of mouse bone collagenase activity in the presence of serum protein by addition of trypsin. Proc. Soc exp. Biol. (N.Y.)139, 1038–1041 (1972).

    Google Scholar 

  21. Sakamoto, S., Goldhaber, P., Climcher, M. J.: The further purification and characterization of mouse bone collagenase. Calc. Tiss. Res.10, 142–151 (1972).

    Google Scholar 

  22. Shimizu, M., Glimcher, M. J., Travis, D. F., Goldhaber, P.: Mouse bone collagenase: Isolation, partial purfication and mechanism of action. Proc. Soc. exp. Biol. (N.Y.)130, 1175–1180 (1969).

    Google Scholar 

  23. Vaes, G.: A latent collagenase released by bone and skin explants in culture. Biochem. J.123, 23 (1971).

    Google Scholar 

  24. Vogel, R., Trautschold, J., Werle, E.: Inhibitors from blood serum. Natural proteinase inhibitors, p. 57. New York: Academic Press 1968.

    Google Scholar 

  25. Wu, Feng Chi, Laskowski, M.: Crystalline acid-labile trypsin inhibitor from bovine blood plasma. J. biol. Chem.235, 1680–1685 (1960).

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Oral Histopathology and Periodontology, Harvard School of Dental Medicine, and Children's Hospital Medical Center, 300 Longwood Avenue, 02115, Boston, Massachusetts, USA

    S. Sakamoto, S. Goldhaber & M. J. Glimcher M.D.

  2. Department of Orthopedic Surgery, Harvard Medical School, and Children's Hospital Medical Center, 300 Longwood Avenue, 02115, Boston, Massachusetts, USA

    S. Sakamoto, S. Goldhaber & M. J. Glimcher M.D.

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  1. S. Sakamoto
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  2. S. Goldhaber
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  3. M. J. Glimcher M.D.
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Additional information

This investigation was supported in part by grants from the United States Public Health Service, National Institutes of Health (DE-2849 and AM 15671).

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Sakamoto, S., Goldhaber, S. & Glimcher, M.J. Further studies on the nature of the components in serum which inhibit mouse bone collagenase. Calc. Tis Res. 10, 280–288 (1972). https://doi.org/10.1007/BF02012559

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  • DOI: https://doi.org/10.1007/BF02012559

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