ANIMAL COLLAGENASE AND COLLAGEN METABOLISM 685 



dishes on filter paper in Tyrode solution, collagenase could be har- 

 vested from the culture medium (Nagai et al., 1963). After several 

 days of incubation, tissue fragments were removed, the breakdown 

 products were dialyzed away, and the fluid in the bag was lyophil- 

 ized to a dry powder. This material has been partially purified on 

 Sephadex columns and compared with bacterial collagenase. Par- 

 tially purified tadpole collagenase has a pH optimum between 6.5 

 and 7.8, is destroyed by heating between 50 and 60 °C for 10 min- 

 utes, and is reversibly inhibited by EDTA. It probably has a lower 

 molecular weight than the Clostridium collagenase as indicated by 

 elution time from Sephadex columns. 



Using the radioactive collagen microassav, we have reexplored the 

 possibility of extracting substantial amounts of collagenase from 

 tadpole tail tissues, by making extracts of control and resorbing tail 

 fin with and without EDTA. (It has been found that the firm en- 

 zyme-substrate complex between bacterial collagenase and solid 

 collagen can be broken with this chelating agent (Gallop et al., 

 1957). The activity of the enzyme can be restored with calcium.) 

 We also carefully reexamined the effect of known proteases on our 

 reconstituted collagen substrate (Table III). We have found a 

 small but definite amount of digestion of reconstituted collagen 

 fibrils by very high concentrations of trypsin, chymotrypsin, papain, 

 elastase, and pronase. This activity had to be differentiated from that 

 of collagenase in order to evaluate the true effect of tissue extracts. 

 It was noted that, unlike that of collagenase, the action of the other 

 proteolytic enzvmes was not progressive with time but was complete 

 within 1 to 2 hours. Moreover, considerably larger amounts of en- 

 zyme were required for detection of this minimal activity, except 

 for pronase, which was effective at lower concentrations. A second 

 significant difference between true collagenolytic action and that of 

 the other enzymes was found in response to EDTA. Both bacterial 

 and tadpole collagenase showed complete reversible inhibition by 

 low concentrations of EDTA, whereas the other proteases were only 

 slightly affected (Table III). By tabulating the fractional inhibition 

 caused by EDTA (last column. Table III) we have a sensitive index 

 whereby collagenase activity may be differentiated from nonspecific 



