Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1985 Nov 1;231(3):505–510. doi: 10.1042/bj2310505

Purification of a metalloproteinase inhibitor from human rheumatoid synovial fluid.

E Mercer, T E Cawston, M de Silva, B L Hazleman
PMCID: PMC1152779  PMID: 3000352

Abstract

A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A-Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases ('TIMP') recently purified from connective-tissue culture medium.

Full text

PDF
503

Images in this article

508Image
on p.508

Selected References

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

  1. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Bunning R. A., Murphy G., Kumar S., Phillips P., Reynolds J. J. Metalloproteinase inhibitors from bovine cartilage and body fluids. Eur J Biochem. 1984 Feb 15;139(1):75–80. doi: 10.1111/j.1432-1033.1984.tb07978.x. [DOI] [PubMed] [Google Scholar]
  4. Cawston T. E., Barrett A. J. A rapid and reproducible assay for collagenase using [1-14C]acetylated collagen. Anal Biochem. 1979 Nov 1;99(2):340–345. doi: 10.1016/s0003-2697(79)80017-2. [DOI] [PubMed] [Google Scholar]
  5. Cawston T. E., Galloway W. A., Mercer E., Murphy G., Reynolds J. J. Purification of rabbit bone inhibitor of collagenase. Biochem J. 1981 Apr 1;195(1):159–165. doi: 10.1042/bj1950159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cawston T. E., Mercer E., Tyler J. A. The activation of latent pig synovial collagenase. Biochim Biophys Acta. 1981 Jan 15;657(1):73–83. doi: 10.1016/0005-2744(81)90131-5. [DOI] [PubMed] [Google Scholar]
  7. Cawston T. E., Mercer E., de Silva M., Hazleman B. L. Metalloproteinases and collagenase inhibitors in rheumatoid synovial fluid. Arthritis Rheum. 1984 Mar;27(3):285–290. doi: 10.1002/art.1780270306. [DOI] [PubMed] [Google Scholar]
  8. Cawston T. E., Murphy G., Mercer E., Galloway W. A., Hazleman B. L., Reynolds J. J. The interaction of purified rabbit bone collagenase with purified rabbit bone metalloproteinase inhibitor. Biochem J. 1983 May 1;211(2):313–318. doi: 10.1042/bj2110313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cawston T. E., Tyler J. A. Purification of pig synovial collagenase to high specific activity. Biochem J. 1979 Dec 1;183(3):647–656. doi: 10.1042/bj1830647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dean D. D., Woessner J. F., Jr Extracts of human articular cartilage contain an inhibitor of tissue metalloproteinases. Biochem J. 1984 Feb 15;218(1):277–280. doi: 10.1042/bj2180277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  12. Murphy G., Cartwright E. C., Sellers A., Reynolds J. J. The detection and characterisation of collagenase inhibitors from rabbit tissues in culture. Biochim Biophys Acta. 1977 Aug 11;483(2):493–498. doi: 10.1016/0005-2744(77)90080-8. [DOI] [PubMed] [Google Scholar]
  13. Murphy G., Cawston T. E., Reynolds J. J. An inhibitor of collagenase from human amniotic fluid. Purification, characterization and action on metalloproteinases. Biochem J. 1981 Apr 1;195(1):167–170. doi: 10.1042/bj1950167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Murphy G., Werb Z. Tissue inhibitor of metalloproteinases. Identification of precursor forms synthesized by human fibroblasts in culture. Biochim Biophys Acta. 1985 Apr 17;839(2):214–218. doi: 10.1016/0304-4165(85)90039-x. [DOI] [PubMed] [Google Scholar]
  15. Sakamoto S., Sakamoto M., Matsumoto A., Goldhaber P., Glimcher M. J. Latent collagenase from the culture medium of embryonic chick bones. FEBS Lett. 1978 Apr 1;88(1):53–58. doi: 10.1016/0014-5793(78)80605-x. [DOI] [PubMed] [Google Scholar]
  16. Sakamoto S., Sakamoto M., Matsumoto A., Nagayama M., Glimcher M. J. Chick bone collagenase inhibitor and latency of collagenase. Biochem Biophys Res Commun. 1981 Nov 16;103(1):339–346. doi: 10.1016/0006-291x(81)91698-3. [DOI] [PubMed] [Google Scholar]
  17. Sellers A., Reynolds J. J., Meikle M. C. Neutral metallo-proteinases of rabbit bone. Separation in latent forms of distinct enzymes that when activated degrade collagen, gelatin and proteoglycans. Biochem J. 1978 May 1;171(2):493–496. doi: 10.1042/bj1710493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stricklin G. P., Bauer E. A., Jeffrey J. J., Eisen A. Z. Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures. Biochemistry. 1977 Apr 19;16(8):1607–1615. doi: 10.1021/bi00627a013. [DOI] [PubMed] [Google Scholar]
  19. Stricklin G. P., Welgus H. G. Human skin fibroblast collagenase inhibitor. Purification and biochemical characterization. J Biol Chem. 1983 Oct 25;258(20):12252–12258. [PubMed] [Google Scholar]
  20. Vater C. A., Mainardi C. L., Harris E. D., Jr Inhibitor of human collagenase from cultures of human tendon. J Biol Chem. 1979 Apr 25;254(8):3045–3053. [PubMed] [Google Scholar]
  21. Welgus H. G., Stricklin G. P. Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid. J Biol Chem. 1983 Oct 25;258(20):12259–12264. [PubMed] [Google Scholar]
  22. Woolley D. E., Roberts D. R., Evanson J. M. Small molecular weight beta 1 serum protein which specifically inhibits human collagenases. Nature. 1976 May 27;261(5558):325–327. doi: 10.1038/261325a0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES