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The further purification and characterization of mouse bone collagenase

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Abstract

Mouse bone collagenase, a specific tissue collagenase, was isolated from the tissue culture media of 5 day old mouse tibiae. By a combination of (NH4)2SO4 precipitation, molecular sieve filtration and ion exchange chromatography a fraction was obtained with a yield of approximately 11% which had a specific enzyme activity 120-fold greater than the original crude extract. The molecular weight of the fraction containing the enzyme activity was approximately 41000 as determined by calibrated molecular sieve filtration studies. There were at least two distinct major fractions and one minor fraction possessing collagenase activity which had distinct isoelectric points. It is not clear whether these fractions represent isoenzymes from bone or from bone and cartilage, or are derived from a single enzyme which has been minimally degraded by the extraction and purification techniques used. Collagenase activity was inhibited by EDTA, cysteine and horse serum, but not by phenylmethylsulfonylfluoride, epsilon amino caproic acid or soybean trypsin inhibitor.

Résumé

De la collagénase osseuse de souris, une collagénase tissulaire spécifique, est isolée du milieu de culture tissulaire d'un tibia de souris de 5 jours. En combinant une précipitation de (NH4)2SO4, une filtration moléculaire sur tamis et la chromatographie par échangeurs d'ions, on obtient une fraction d'un rendement d'environ 11%, qui possède une activité enzymatique spécifique 120 fois plus élevée que l'extrait original total. Le poids moléculaire de la fraction, contenant l'activité enzymatique, est d'environ 41000, en se basant sur des études de filtration par tamis moléculaire calibré. Il existe au moins deux fractions principales distinctes et une fraction plus faible, ayant des activités en collagénase et des points isoélectriques distincts. Il n'est pas démontré que ces fractions constituent des isoenzymes de l'os ou de l'os et du cartilage, ou sont dérivés d'une seule enzyme, peu dégradée par l'extraction et la purification. L'activité de la collagénase est inhibée par l'EDTA, la cystéine, le sérum de cheval; mais elle résiste au fluorure de phénylméthyle-sulfonyle, à l'acide epsilon aminocaproique et à l'inhibiteur de trypsine de graine de soja.

Zusammenfassung

Mäuseknochen-Kollagenase, eine spezifische Gewebe-Kollagenase, wurde aus Gewebekulturmedien von 5 Tage alten Mäusetibiae isoliert. Durch eine Kombination von (NH4)2SO4-Ausfällung, Gelfiltration und Ionenaustausch-Chromatographie wurde mit ungefähr 11% Ausbeute eine Fraktion erhalten, die eine spezifische Enzymaktivität besaß, welche 120mal größer war als diejenige des ursprünglichen unbehandelten Extraktes. Das Molekulargewicht der die Enzymaktivität enthaltenden Fraktion war ungefähr 41000, was durch kalibrierte Gelfiltrations-Untersuchungen bestimmt wurde. Mindestens zwei verschiedene Hauptfraktionen und eine kleinere Fraktion mit Kollagenaseaktivität wurden erhalten, welche verschiedene isoelektrische Punkte hatten. Es ist unklar, ob diese Fraktionen Isoenzyme aus Knochen oder aus Knochen und Knorpel darstellen, oder ob sie von einem einzelnen Enzym stammen, welches durch die verwendeten Extraktions- und Reinigungstechniken minimal degradiert wurde. Die Kollagenaseaktivität wurde durch EDTA, Cystein und Pferdeserum gehemmt, nicht aber durch Phenylmethylsulfonylfluorid, Epsilon-amino-capronsäure oder Soyabohnen-Trypsin-Hemmer.

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Authors and Affiliations

  1. Department of Oral Histopathology and Periodontology, Harvard School of Dental Medicine, Children's Hospital Medical Center, 300 Longwood Avenue, 02115, Boston, Massachusetts, USA

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

  2. Department of Orthopedic Surgery, Harvard Medical School, Children's Hospital Medical Center, 300 Longwood Avenue, 02115, Boston, Massachusetts, USA

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

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  1. S. Sakamoto
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  2. P. Goldhaber
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  3. M. J. Glimcher
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Sakamoto, S., Goldhaber, P. & Glimcher, M.J. The further purification and characterization of mouse bone collagenase. Calc. Tis Res. 10, 142–151 (1972). https://doi.org/10.1007/BF02012544

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