28 Ch. M. Lapiere 



With G. Vaes, we have been able to demonstrate some collagenolytic activity in 

 homogenates of bone from calvaria of new born rats. This enzymatic activity is also 

 increased by treatment with parathormone. We are, however, not yet able to 

 correlate these results with the activity of a collagenase-like enzyme. Woods and 

 Nichols (1963, 1964) have demonstrated a similar enzymatic system in homogenates 

 of bone cells. 



The only isolated animal coUagenase is the enzyme collected from tissue culture 

 of tadpole skin (Lapiere and Gross, 1963). This enzyme is now purrified (Nagai 

 et al., 1963) and characterized (Nagai et ai, 1964). It possesses all the characteristics 

 required for being a true collagenase. It lyses the native collagen at a constant rate 

 under physiological conditions. Its activity on the collagen molecule is very specific 

 and limited. 



Although this enzyme is present in tissue culture we have never been able to 

 demonstrate its presence in living tissues. On the other hand we found a slight 

 amount of protease-like activity on native collagen (Lapiere and Gross, 1963). 

 Because of these findings we became very careful about the conditions under which 

 the enzyme assay has to be carried out in order to detect a collagenase-like enzyme 

 in the presence of proteolytic enzymes. 



The enzyme has to be active on native collagen. Denaturation to gelatin occurs in 

 certain circumstances. There are certain limits of pH and temperature outside of 

 which the collagen does not remain native. This is demonstrated by an experiment in 

 which different samples of radioactive highly purified skin collagen (either in fibrous 

 form or in solution) are placed for several hours at 37 °C in buffered solution of pH 

 ranging between 2.8 and 9.4. After bringing the pH back to 7.5 the activity of tryp- 

 sin is used to check the native or denatured state of the collagen. A small and rather 

 constant amount of dialysable labelled collagen fragments are liberated from the 

 samples preincubated between limits of pH ranging from 4.8 to 9.1. Below and above 

 these limits, collagen is degraded to gelatin and therefore becomes susceptible to the 

 proteolytic activity of trypsin. 



Assaying fractionated bone extract on native collagen we found collagenolytic 

 activity at pH 5.5 and 7.0 both in the non particulate part of the homogenate and in 

 the light and heavy particles. However we do not feel that this activity is collagenase- 

 like because it does not fulfill all the conditions of enzyme kinetics in the same way 

 as the bacterial or the tadpole collagenase. 



We know that bacterial collagenase lyses collagen at a fast rate while trypsin at 

 pH 7.0 and pepsin at pH 5.5 have only very limited activity (Fig. 3). When we study 

 the regression lines of concentration or time dependence activity of these three 

 enzymes we can see that none of them would pass through the origin. This may mean 

 that the substrate, the native collagen in its fibrous form, is not homogeneous. A small 

 part of it (5 — 12Vo) is more susceptible to enzyme attack. It could represent a por- 

 tion of the collagen which is not organized in dense fibers. The activity of trypsin on 

 native collagen is indeed much more significant (80"/o) when the collagen is in solu- 

 tion. With respect to these considerations we have to accept that we can only call by 

 the name "collagenase" an enzyme displaying sufficient activity on native collagen 

 whatever its stage of aggregation might be. As far as I know, none of the works 

 published until now has given evidence of such an enzyme in homogenates of bone or 

 of any other tissues. 



