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. 1975 Oct;152(1):131–142. doi: 10.1042/bj1520131

Purification and characterization of a collagenase extracted from rabbit tumours.

P A McCroskery, J F Richards, E D Harris Jr
PMCID: PMC1172448  PMID: 2161

Abstract

A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.

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Selected References

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

  1. Abe S., Nagai Y. Evidence for the presence of a complex of collagenase with alpha2-macroglobulin in human rheumatoid synovial fluid: a possible regulatory mechanism of collagenase activity in vivo. J Biochem. 1973 Apr;73(4):897–900. doi: 10.1093/oxfordjournals.jbchem.a130153. [DOI] [PubMed] [Google Scholar]
  2. BONTING S. L., CARAVAGGIO L. L., HAWKINS N. M. Studies on sodium-potassium-activated adenosinetriphosphatase. VI. Its role in cation transport in the lens of cat, calf and rabbit. Arch Biochem Biophys. 1963 Apr;101:47–55. doi: 10.1016/0003-9861(63)90532-0. [DOI] [PubMed] [Google Scholar]
  3. Barrett A. J., Starkey P. M. The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J. 1973 Aug;133(4):709–724. doi: 10.1042/bj1330709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bauer E. A., Eisen A. Z., Jeffrey J. J. Studies on purified rheumatoid synovial collagenase in vitro and in vivo. J Clin Invest. 1971 Oct;50(10):2056–2064. doi: 10.1172/JCI106699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berman M. B., Manabe R. Corneal collagenases: evidence for zinc metalloenzymes. Ann Ophthalmol. 1973 Nov;5(11):1193–passim. [PubMed] [Google Scholar]
  6. Chung E., Miller E. J. Collagen polymorphism: characterization of molecules with the chain composition (alpha 1 (3)03 in human tissues. Science. 1974 Mar;183(130):1200–1201. doi: 10.1126/science.183.4130.1200. [DOI] [PubMed] [Google Scholar]
  7. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [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 activity in vivo and in vitro. Proc Natl Acad Sci U S A. 1971 Jan;68(1):248–251. doi: 10.1073/pnas.68.1.248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Evanson J. M., Jeffrey J. J., Krane S. M. Human collagenase: identification and characterization of an enzyme from rheumatoid synovium in culture. Science. 1967 Oct 27;158(3800):499–502. doi: 10.1126/science.158.3800.499. [DOI] [PubMed] [Google Scholar]
  10. Fietzek P. P., Rexrodt F. W., Hopper K. E., Kühn K. The covalent structure of collagen. 2. The amino-acid sequence of alpha1-CB7 from calf-skin collagen. Eur J Biochem. 1973 Oct 5;38(2):396–400. doi: 10.1111/j.1432-1033.1973.tb03072.x. [DOI] [PubMed] [Google Scholar]
  11. Fullmer H. M., Taylor R. E., Guthrie R. W. Human gingival collagenase: purification, molecular weight, and inhibitor studies. J Dent Res. 1972 Mar-Apr;51(2):349–355. doi: 10.1177/00220345720510022101. [DOI] [PubMed] [Google Scholar]
  12. GLIMCHER M. J., FRANCOIS C. J., RICHARDS L., KRANE S. M. THE PRESENCE OF ORGANIC PHOSPHORUS IN COLLAGENS AND GELATINS. Biochim Biophys Acta. 1964 Dec 9;93:585–602. doi: 10.1016/0304-4165(64)90342-3. [DOI] [PubMed] [Google Scholar]
  13. GROSS J., KIRK D. The heat precipitation of collagen from neutral salt solutions: some rate-regulating factors. J Biol Chem. 1958 Aug;233(2):355–360. [PubMed] [Google Scholar]
  14. GROSS J. [Studies on the formation of collagen. III. Time-dependent solubility changes of collagen in vitro]. J Exp Med. 1958 Aug 1;108(2):215–226. doi: 10.1084/jem.108.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gross J., Harper E., Harris E. D., McCroskery P. A., Highberger J. H., Corbett C., Kang A. H. Animal collagenases: specificity of action, and structures of the substrate cleavage site. Biochem Biophys Res Commun. 1974 Nov 27;61(2):605–612. doi: 10.1016/0006-291x(74)91000-6. [DOI] [PubMed] [Google Scholar]
  16. HEPPEL L. A., HILMORE R. J. Purification and properties of 5-nucleotidase. J Biol Chem. 1951 Feb;188(2):665–676. [PubMed] [Google Scholar]
  17. Harpel P. C. Studies on human plasma alpha 2-macroglobulin-enzyme interactions. Evidence for proteolytic modification of the subunit chain structure. J Exp Med. 1973 Sep 1;138(3):508–521. doi: 10.1084/jem.138.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  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, Farrell M. E. Resistance to collagenase: a characteristic of collagen fibrils cross-linked by formaldehyde. Biochim Biophys Acta. 1972 Aug 31;278(1):133–141. doi: 10.1016/0005-2795(72)90114-6. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Harris E. D., Jr, Krane S. M. Collagenases (first of three parts). N Engl J Med. 1974 Sep 12;291(11):557–563. doi: 10.1056/NEJM197409122911105. [DOI] [PubMed] [Google Scholar]
  24. Harris E. D., Jr, Krane S. M. Collagenases (second of three parts). N Engl J Med. 1974 Sep 19;291(12):605–609. doi: 10.1056/NEJM197409192911205. [DOI] [PubMed] [Google Scholar]
  25. Harris E. D., Jr, Krane S. M. Collagenases (third of three parts). N Engl J Med. 1974 Sep 26;291(13):652–661. doi: 10.1056/NEJM197409262911305. [DOI] [PubMed] [Google Scholar]
  26. Kruze D., Wojtecka E. Activation of leucocyte collagenase proenzyme by rheumatoid synovial fluid. Biochim Biophys Acta. 1972 Dec 28;285(2):436–446. doi: 10.1016/0005-2795(72)90330-3. [DOI] [PubMed] [Google Scholar]
  27. LEWIS M. S., PIEZ K. A. SEDIMENTATION-EQUILIBRIUM STUDIES OF THE MOLECULAR WEIGHT OF SINGLE AND DOUBLE CHAINS FROM RAT-SKIN COLLAGEN. Biochemistry. 1964 Aug;3:1126–1131. doi: 10.1021/bi00896a020. [DOI] [PubMed] [Google Scholar]
  28. LOWRY O. H., ROBERTS N. R., WU M. L., HIXON W. S., CRAWFORD E. J. The quantitative histochemistry of brain. II. Enzyme measurements. J Biol Chem. 1954 Mar;207(1):19–37. [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. MORTON R. K. Alkaline phosphatase of milk. 2. Purification of the enzyme. Biochem J. 1953 Dec;55(5):795–800. doi: 10.1042/bj0550795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McCroskery P. A., Wood S., Jr, Harris E. D., Jr Gelatin: a poor substrate for a mammalian collegenase. Science. 1973 Oct 5;182(4107):70–71. doi: 10.1126/science.182.4107.70. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. NORDLIE R. C., ARION W. J. EVIDENCE FOR THE COMMON IDENTITY OF GLUCOSE 6-PHOSPHATASE, INORGANIC PYROPHOSPHATASE, AND PYROPHOSPHATE-GLUCOSE PHOSPHOTRANSFERASE. J Biol Chem. 1964 Jun;239:1680–1685. [PubMed] [Google Scholar]
  35. OUCHTERLONY O. Diffusion-in-gel methods for immunological analysis. II. Prog Allergy. 1962;6:30–154. doi: 10.1159/000313795. [DOI] [PubMed] [Google Scholar]
  36. Oronsky A. L., Perper R. J., Schroder H. C. Phagocytic release and activation of human leukocyte procollagenase. Nature. 1973 Dec 14;246(5433):417–419. doi: 10.1038/246417a0. [DOI] [PubMed] [Google Scholar]
  37. PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. PROCKOP D. J., UDENFRIEND S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem. 1960 Nov;1:228–239. doi: 10.1016/0003-2697(60)90050-6. [DOI] [PubMed] [Google Scholar]
  39. Sakai T., Gross J. Some properties of the products of reaction of tadpole collagenase with collagen. Biochemistry. 1967 Feb;6(2):518–528. doi: 10.1021/bi00854a021. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Sakamoto S., Sakamoto M., Goldhaber P., Glimcher M. J. The inhibition of mouse bone collagenase by lysozyme. Calcif Tissue Res. 1974 May 28;14(4):291–299. doi: 10.1007/BF02060303. [DOI] [PubMed] [Google Scholar]
  42. Steers E., Jr, Cuatrecasas P., Pollard H. B. The purification of beta-galactosidase from Escherichia coli by affinity chromatography. J Biol Chem. 1971 Jan 10;246(1):196–200. [PubMed] [Google Scholar]
  43. Traub W., Piez K. A. The chemistry and structure of collagen. Adv Protein Chem. 1971;25:243–352. doi: 10.1016/s0065-3233(08)60281-8. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  46. Werb Z., Burleigh M. C. A specific collagenase from rabbit fibroblasts in monolayer culture. Biochem J. 1974 Feb;137(2):373–385. doi: 10.1042/bj1370373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Werb Z., Burleigh M. C., Barrett A. J., Starkey P. M. The interaction of alpha2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases. Biochem J. 1974 May;139(2):359–368. doi: 10.1042/bj1390359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Woessner J. F., Jr, Ryan J. N. Collagenase activity in homogenates of the involuting rat uterus. Biochim Biophys Acta. 1973 Jun 6;309(2):397–405. doi: 10.1016/0005-2744(73)90038-7. [DOI] [PubMed] [Google Scholar]

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