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. 1974 Feb;137(2):373–385. doi: 10.1042/bj1370373

A specific collagenase from rabbit fibroblasts in monolayer culture

Zena Werb 1, Mary C Burleigh 1
PMCID: PMC1166126  PMID: 4363113

Abstract

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24°C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH2Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bainton D. F. Sequential degranulation of the two types of polymorphonuclear leukocyte granules during phagocytosis of microorganisms. J Cell Biol. 1973 Aug;58(2):249–264. doi: 10.1083/jcb.58.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrett A. J. Cathepsin D. Purification of isoenzymes from human and chicken liver. Biochem J. 1970 Apr;117(3):601–607. doi: 10.1042/bj1170601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barrett A. J., Dingle J. T. The inhibition of tissue acid proteinases by pepstatin. Biochem J. 1972 Apr;127(2):439–441. doi: 10.1042/bj1270439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barrett A. J. Human cathepsin B1. Purification and some properties of the enzyme. Biochem J. 1973 Apr;131(4):809–822. doi: 10.1042/bj1310809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burleigh M. C., Barrett A. J., Lazarus G. S. Cathepsin B1. A lysosomal enzyme that degrades native collagen. Biochem J. 1974 Feb;137(2):387–398. doi: 10.1042/bj1370387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Donoff R. B., McLennan J. E., Grillo H. C. Preparation and properties of collagenases from epithelium and mesenchyme of healing mammalian wounds. Biochim Biophys Acta. 1971 Mar 10;227(3):639–653. doi: 10.1016/0005-2744(71)90014-3. [DOI] [PubMed] [Google Scholar]
  8. Eisen A. Z., Bauer E. A., Jeffrey J. J. Animal and human collagenases. J Invest Dermatol. 1970 Dec;55(6):359–373. doi: 10.1111/1523-1747.ep12260483. [DOI] [PubMed] [Google Scholar]
  9. Eisen A. Z., Jeffrey J. J., Gross J. Human skin collagenase. Isolation and mechanism of attack on the collagen molecule. Biochim Biophys Acta. 1968 Mar 25;151(3):637–645. doi: 10.1016/0005-2744(68)90010-7. [DOI] [PubMed] [Google Scholar]
  10. Evanson J. M., Jeffrey J. J., Krane S. M. Studies on collagenase from rheumatoid synovium in tissue culture. J Clin Invest. 1968 Dec;47(12):2639–2651. doi: 10.1172/JCI105947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. GROSS J., LAPIERE C. M. Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci U S A. 1962 Jun 15;48:1014–1022. doi: 10.1073/pnas.48.6.1014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gross J., Nagai Y. Specific degradation of the collagen molecule by tadpole collagenolytic enzyme. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1197–1204. doi: 10.1073/pnas.54.4.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. HARTLEY B. S. Proteolytic enzymes. Annu Rev Biochem. 1960;29:45–72. doi: 10.1146/annurev.bi.29.070160.000401. [DOI] [PubMed] [Google Scholar]
  14. Hamerman D., Janis R., Smith C. Cartilage matrix depletion by rheumatoid synovial cells in tissue culture. J Exp Med. 1967 Dec 1;126(6):1005–1012. doi: 10.1084/jem.126.6.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harper E., Bloch K. J., Gross J. The zymogen of tadpole collagenase. Biochemistry. 1971 Aug 3;10(16):3035–3041. doi: 10.1021/bi00792a008. [DOI] [PubMed] [Google Scholar]
  16. Harper E., Gross J. Collagenase, procollagenase and activator relationships in tadpole tissue cultures. Biochem Biophys Res Commun. 1972 Sep 5;48(5):1147–1152. doi: 10.1016/0006-291x(72)90830-3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Harris E. D., Jr, DiBona D. R., Krane S. M. Collagenases in human synovial fluid. J Clin Invest. 1969 Nov;48(11):2104–2113. doi: 10.1172/JCI106177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harris E. D., Jr, Faulkner C. S., 2nd, Wood S., Jr Collagenase in carcinoma cells. Biochem Biophys Res Commun. 1972 Sep 5;48(5):1247–1253. doi: 10.1016/0006-291x(72)90845-5. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Harris E. D., Jr, Krane S. M. Effects of colchicine on collagenase in cultures of rheumatoid synovium. Arthritis Rheum. 1971 Nov-Dec;14(6):669–684. doi: 10.1002/art.1780140602. [DOI] [PubMed] [Google Scholar]
  23. Henson P. M. The immunologic release of constituents from neutrophil leukocytes. II. Mechanisms of release during phagocytosis, and adherence to nonphagocytosable surfaces. J Immunol. 1971 Dec;107(6):1547–1557. [PubMed] [Google Scholar]
  24. Hook C. W., Brown S. I., Iwanij W., Nakanishi I. Characterization and inhibition of corneal collagenase. Invest Ophthalmol. 1971 Jul;10(7):496–503. [PubMed] [Google Scholar]
  25. Jeffrey J. J., Gross J. Collagenase from rat uterus. Isolation and partial characterization. Biochemistry. 1970 Jan 20;9(2):268–273. doi: 10.1021/bi00804a012. [DOI] [PubMed] [Google Scholar]
  26. Kang A. H., Nagai Y., Piez K. A., Gross J. Studies on the structure of collagen utilizing a collagenolytic enzyme from tadpole. Biochemistry. 1966 Feb;5(2):509–515. doi: 10.1021/bi00866a016. [DOI] [PubMed] [Google Scholar]
  27. Lazarus G. S., Brown R. S., Daniels J. R., Fullmer H. M. Human granulocyte collagenase. Science. 1968 Mar 29;159(3822):1483–1485. doi: 10.1126/science.159.3822.1483. [DOI] [PubMed] [Google Scholar]
  28. Lazarus G. S., Daniels J. R., Lian J., Burleigh M. C. Role of granulocyte collagenase in collagen degradation. Am J Pathol. 1972 Sep;68(3):565–578. [PMC free article] [PubMed] [Google Scholar]
  29. Lazarus G. S., Fullmer H. M. Collagenase production by human dermis in vitro. J Invest Dermatol. 1969 Jun;52(6):545–547. doi: 10.1038/jid.1969.94. [DOI] [PubMed] [Google Scholar]
  30. NAGAI Y., GROSS J., PIEZ K. A. DISC ELECTROPHORESIS OF COLLAGEN COMPONENTS. Ann N Y Acad Sci. 1964 Dec 28;121:494–500. doi: 10.1111/j.1749-6632.1964.tb14221.x. [DOI] [PubMed] [Google Scholar]
  31. Nagai Y., Hori H. Vertebrate collagenase: direct extraction from animal skin and human synovial membrane. J Biochem. 1972 Nov;72(5):1147–1153. doi: 10.1093/oxfordjournals.jbchem.a130002. [DOI] [PubMed] [Google Scholar]
  32. Nagai Y., Lapiere C. M., Gross J. Tadpole collagenase. Preparation and purification. Biochemistry. 1966 Oct;5(10):3123–3130. doi: 10.1021/bi00874a007. [DOI] [PubMed] [Google Scholar]
  33. Poole A. R., Dingle J. T., Barrett A. J. The immunocytochemical demonstration of cathepsin D. J Histochem Cytochem. 1972 Apr;20(4):261–265. doi: 10.1177/20.4.261. [DOI] [PubMed] [Google Scholar]
  34. Powers J. C., Tuhy P. M. Active-site specific inhibitors of elastase. J Am Chem Soc. 1972 Sep 6;94(8):6544–6545. doi: 10.1021/ja00773a049. [DOI] [PubMed] [Google Scholar]
  35. Pérez-Tamayo R. Collagen resorption in carrageenin granulomas. I. Collagenolytic activity in in vitro explants. Lab Invest. 1970 Feb;22(2):137–141. [PubMed] [Google Scholar]
  36. Reynolds J. M. Motivating office personnel for increased efficiency. Trans Eur Orthod Soc. 1973:379–385. [PubMed] [Google Scholar]
  37. Robertson P. B., Ryel R. B., Taylor R. E., Shyu K. W., Fullmer H. M. Collagenase: localization in polymorphonuclear leukocyte granules in the rabbit. Science. 1972 Jul 7;177(4043):64–65. doi: 10.1126/science.177.4043.64. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Tokoro Y., Eisen A. Z., Jeffrey J. J. Characterization of a collagenase from rat skin. Biochim Biophys Acta. 1972 Jan 20;258(1):289–302. doi: 10.1016/0005-2744(72)90986-2. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. WUENSCH E., HEIDRICH H. G. ZUR QUANTITATIVEN BESTIMMUNG DER KOLLAGENASE. Hoppe Seylers Z Physiol Chem. 1963;333:149–151. doi: 10.1515/bchm2.1963.333.1.149. [DOI] [PubMed] [Google Scholar]
  42. ZUCKER-FRANKLIN D., HIRSCH J. G. ELECTRON MICROSCOPE STUDIES ON THE DEGRANULATION OF RABBIT PERITONEAL LEUKOCYTES DURING PHAGOCYTOSIS. J Exp Med. 1964 Oct 1;120:569–576. doi: 10.1084/jem.120.4.569. [DOI] [PMC free article] [PubMed] [Google Scholar]

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