680 C. M. LAPIERE AND J. GROSS 



Enzymes have been isolated from mammalian white blood cells 

 (Sherry et ah, 1954) and from liver lysosomal fractions (Franklin 

 and Wynn, 1961) and uterus (Morrione and Seifter, 1962; Woessner, 

 1962 ) which attack collagen to a slight extent in the acid pH range. 

 A type of collagenolvtic activity in certain pancreatic extracts has 

 been claimed ( Houck and Patol, 1962 ) . The only well characterized, 

 true collagenase has been isolated from species of Clostridium 

 (Mandl, 1961). 



In considering the possible reasons for failure to detect an animal 

 collagenase, we suspected that such an enzyme must be present in 

 extremely low concentrations in highly localized regions, otherwise 

 uncontrolled degradation incompatible with functional stability 

 would occur. It seemed unlikely that any of the current assay pro- 

 cedures would be sensitive enough to detect low concentrations of 

 this enzyme. In addition, it has been established (Gallop et al., 

 1957) that bacterial collagenase binds to collagen fibers and is re- 

 leased only after digesting the protein; this being the case, the usual 

 extraction procedures might fail to release animal collagenase from 

 the collagen still present in the tissues. It also seemed possible that 

 tissue collagenase might have a very short active life and either be 

 rapidly inhibited or be degraded by other enzymes. 



For these reasons we set out to detect collagenase activity by 

 culturing small bits of tissue on a reconstituted fibrous collagen 

 substrate. It was thought that any newly produced or activated 

 collagenase might diffuse away from the tissue, bind to the fibrils 

 of the substrate, digest them, and thereby reveal activity as an 

 expanding area of lysis. This prediction was borne out and we have 

 found collagenolytic activity in cultured tadpole tissues and in mam- 

 malian uterus and bone (Gross and Lapiere, 1962; Gross, Lapiere, 

 and Tanzer, 1963). Figure 11 presents photographs of a typical 

 culture of a collagen gel, showing the expanding areas of lysis 

 around the explants of tail fin tissue. 



The collagen substrates used were either acid-extracted calf skin 

 collagen or saline-extracted radioactive guinea pig skin collagen 

 which were purified, lyophilized, and dissolved in cold physiologic 

 salt solutions in concentrations of approximately 0.1 per cent. The 

 collagen polymerized to typical cross-striated fibrils (characteristic 



