An assay system for estimating quantitatively collagenase-like activity present in bone cells has been developed as part of a more general investigation of mechanisms of bone resorption. Methods are described for preparing from bone a C14-labeled collagen which is relatively pure and highly resistant to degradation by trypsin although readily broken down by bacterial collagenase. Collagenolytic activity in homogenates of bone cells harvested from rat metaphyseal bone was measured as the number of counts per minute released in ultrafiltrable form from the C14-labeled collagen substrate after 40 minutes' incubation at 37°C and pH 7.3. Using these techniques, the presence of collagenase-like activity in whole bone cell homogenates was confirmed and the heat lability, partial cation dependence, pH optimum, and some other characteristics of the crude material were determined. Moreover, the major portion of the homogenate activity was found in a particulate fraction sedimenting in a centrifugal field between 700 and 15,000 g. The marked enhancement and solubilization of this activity by surface-active agents or freeze-thawing, together with the presence of considerable acid phosphatase activity in the same fraction and its sedimentation characteristics, suggested that it might be contained in lysosomes or similar bodies. The implications of these observations with respect to the physiology of collagen resorption in general and bone resorption in particular are discussed.

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