Mouse bone collagenase. Purification of the enzyme by heparin-substitutes Sepharose 4B affinity chromatography and preparation of specific antibody to the enzyme

Sakamoto, S.; Sakamoto, M.; Goldhaber, P.; Glimcher, M.J.

Archives of Biochemistry and Biophysics 188(2): 438-449

1978


ISSN/ISBN: 0003-9861
PMID: 209752
DOI: 10.1016/s0003-9861(78)80028-9
Accession: 068524180

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Abstract
Heparin-substituted Sepharose 4B gel affinity chromatography used to purify mouse bone collagenase (EC 3.4.24.3) increased its specific activity 1340-fold (2850 units/mg) with an overall yield of 9% from the original pooled tissue culture medium. The purified enzyme moved as a single band and had a MW of 47,000 as determined by analytical disc gel electrophoresis in the presence of 1% sodium dodecyl sulfate. Antiserum to the purified enzyme was produced in rabbits, and its monospecificity was established by immunoelectrophoresis. The .gamma.-globulin fraction of the antiserum, freed of .alpha.2-macroglobulin by gel filtration, inhibited collagenase activity. The antibody was purified approximately 70-fold by immunoadsorption using enzyme-substituted Sepharose 4B gel. The extent of the immunoinhibition of various tissue collagenases by mouse bone collagenase antibody varied depending on the tissue and species from which the enzyme was obtained.