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. 1978 May 15;172(2):261–274. doi: 10.1042/bj1720261

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

G Vaes, Y Eeckhout, G Lenaers-Claeys, C François-Gillet, J E Druetz
PMCID: PMC1185692  PMID: 208518

Abstract

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.

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Selected References

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  1. Birkedal-Hansen H., Cobb C. M., Taylor R. E., Fullmer H. M. Procollagenase from bovine gingiva. Biochim Biophys Acta. 1976 Mar 11;429(1):229–238. doi: 10.1016/0005-2744(76)90046-2. [DOI] [PubMed] [Google Scholar]
  2. Birkedal-Hansen H., Cobb C. M., Taylor R. E., Fullmer H. M. Synthesis and release of procollagenase by cultured fibroblasts. J Biol Chem. 1976 May 25;251(10):3162–3168. [PubMed] [Google Scholar]
  3. Dingle J. T. The secretion of enzymes into the pericellular environment. Philos Trans R Soc Lond B Biol Sci. 1975 Jul 17;271(912):315–324. doi: 10.1098/rstb.1975.0055. [DOI] [PubMed] [Google Scholar]
  4. Eeckhout Y., Vaes G. Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation. Biochem J. 1977 Jul 15;166(1):21–31. doi: 10.1042/bj1660021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eeckhout Y., Vaes G. Proceedings: Activation of the inactive precursor of collagenase by kallikrein and plasmin. Arch Int Physiol Biochim. 1974 Oct;82(4):786–786. [PubMed] [Google Scholar]
  6. FELL H. B., DINGLE J. T. Studies on the mode of action of excess of vitamin A. 6. Lysosomal protease and the degradation of cartilage matrix. Biochem J. 1963 May;87:403–408. doi: 10.1042/bj0870403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gillet C., Eeckhout Y., Vaes G. Purification of procollagenase and collagenase by affinity chromatography on Sepharose-collagen. FEBS Lett. 1977 Feb 15;74(1):126–128. doi: 10.1016/0014-5793(77)80768-0. [DOI] [PubMed] [Google Scholar]
  8. Harper E., Gross J. Separation of collagenase and peptidase activities of tadpole tissues in culture. Biochim Biophys Acta. 1970 Feb 11;198(2):286–292. doi: 10.1016/0005-2744(70)90061-6. [DOI] [PubMed] [Google Scholar]
  9. Harris E. D., Jr A collagenolytic system produced by primary cultures of rheumatoid nodule tissue. J Clin Invest. 1972 Nov;51(11):2973–2976. doi: 10.1172/JCI107122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harris E. D., Jr, Krane S. M. An endopeptidase from rheumatoid synovial tissue culture. Biochim Biophys Acta. 1972 Feb 28;258(2):566–576. doi: 10.1016/0005-2744(72)90249-5. [DOI] [PubMed] [Google Scholar]
  11. Hascall V. C., Sajdera S. W. Proteinpolysaccharide complex from bovine nasal cartilage. The function of glycoprotein in the formation of aggregates. J Biol Chem. 1969 May 10;244(9):2384–2396. [PubMed] [Google Scholar]
  12. Hauser P., Vaes G. Degradation of cartilage proteoglycans by a neutral proteinase secreted by rabbit bone-marrow macrophages in culture. Biochem J. 1978 May 15;172(2):275–284. doi: 10.1042/bj1720275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hauser P., Vaes G. Synthesis and secretion by rabbit bone-marrow macrophages in culture of a neutral proteinase that degrades cartilage proteoglycans [proceedings]. Biochem Soc Trans. 1977;5(4):1091–1093. doi: 10.1042/bst0051091. [DOI] [PubMed] [Google Scholar]
  14. Heinegård D., Hascall V. C. Characterization of chondroitin sulfate isolated from trypsin-chymotrypsin digests of cartilage proteoglycans. Arch Biochem Biophys. 1974 Nov;165(1):427–441. doi: 10.1016/0003-9861(74)90182-9. [DOI] [PubMed] [Google Scholar]
  15. Ignarro L. J., Oronsky A. L., Perper R. J. Breakdown of noncollagenous chondromucoprotein matrix by leukocyte lysosome granule lysates from guinea pig, rabbit, and human. Clin Immunol Immunopathol. 1973 Nov;2(1):36–51. doi: 10.1016/0090-1229(73)90034-2. [DOI] [PubMed] [Google Scholar]
  16. Janoff A. At least three human neutrophil lysosomal proteases are capable of degrading joint connective tissues. Ann N Y Acad Sci. 1975 Jun 13;256:402–408. doi: 10.1111/j.1749-6632.1975.tb36066.x. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Lenaers-Claeys G., Vaes G. Conditions governing the release of collagenase and procollagenase by bone explants in culture: effects of heparin. Arch Int Physiol Biochim. 1976;84(3):634–636. [PubMed] [Google Scholar]
  19. Luscombe M., Phelps C. F. Action of degradative enzymes on the light fraction of bovine septa protein polysaccharide. Biochem J. 1967 Apr;103(1):103–109. doi: 10.1042/bj1030103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mathews M. B. Comparative biochemistry of chondroitin sulphate-proteins of cartilage and notochord. Biochem J. 1971 Nov;125(1):37–46. doi: 10.1042/bj1250037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sakamoto S., Goldhaber P., Glimcher M. J. Mouse bone collagenase. The effect of heparin on the amount of enzyme released in tissue culture and on the activity of the enzyme. Calcif Tissue Res. 1973;12(3):247–258. doi: 10.1007/BF02013739. [DOI] [PubMed] [Google Scholar]
  22. Sakamoto S., Goldhaber P., Glimcher M. J. The further purification and characterization of mouse bone collagenase. Calcif Tissue Res. 1972;10(2):142–151. doi: 10.1007/BF02012544. [DOI] [PubMed] [Google Scholar]
  23. Sakamoto S., Sakamoto M., Goldhaber P., Glimcher M. J. Studies on the interaction between heparin and mouse bone collagenase. Biochim Biophys Acta. 1975 Mar 14;385(1):41–50. doi: 10.1016/0304-4165(75)90072-0. [DOI] [PubMed] [Google Scholar]
  24. Sapolsky A. I., Keiser H., Howell D. S., Woessner J. F., Jr Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH. J Clin Invest. 1976 Oct;58(4):1030–1041. doi: 10.1172/JCI108526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sellers A., Cartwright E., Murphy G., Reynolds J. J. Evidence that latent collagenases are enzyme-inhibitor complexes. Biochem J. 1977 May 1;163(2):303–307. doi: 10.1042/bj1630303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shimizu M., Glimcher M. J., Travis D., Goldhaber P. Mouse bone collagenase: isolation, partial purification, and mechanism of action. Proc Soc Exp Biol Med. 1969 Apr;130(4):1175–1180. doi: 10.3181/00379727-130-33747. [DOI] [PubMed] [Google Scholar]
  27. Sopata I., Dancewicz A. M. Presence of a gelatin-specific proteinase and its latent form in human leucocytes. Biochim Biophys Acta. 1974 Dec 29;370(2):510–523. doi: 10.1016/0005-2744(74)90112-0. [DOI] [PubMed] [Google Scholar]
  28. Vaes G., Eeckhout Y., Druetz J. E. A latent neutral protease released by bone in culture. Arch Int Physiol Biochim. 1976;84(3):666–668. [PubMed] [Google Scholar]
  29. Vaes G. Multiple steps in the activation of the inactive precursor of bone collagenase by trypsin. FEBS Lett. 1972 Dec 1;28(2):198–200. doi: 10.1016/0014-5793(72)80711-7. [DOI] [PubMed] [Google Scholar]
  30. Vaes G. On the mechanisms of bone resorption. The action of parathyroid hormone on the excretion and synthesis of lysosomal enzymes and on the extracellular release of acid by bone cells. J Cell Biol. 1968 Dec;39(3):676–697. doi: 10.1083/jcb.39.3.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vaes G. The release of collagenase as an inactive proenzyme by bone explants in culture. Biochem J. 1972 Jan;126(2):275–289. doi: 10.1042/bj1260275. [DOI] [PMC free article] [PubMed] [Google Scholar]

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