682 C. M. LAPIERE AND J. GROSS 



of native structure) when warmed for short periods at 37°C (Gross 

 and Kirk, 1958). These opalescent fibrous gels were resistant to 

 attack by the common proteolytic enzymes (Gross and Lapiere, 

 1962). The tissues of tadpoles (previously sterilized by adding 

 penicillin-streptomycin and chloramphenicol to the aquarium water ) 

 were cultured on sterile collagen gels in small ring chambers or petri 

 dishes using a medium composed of amino acid- and vitamin- 

 supplemented amphibian Tvrode solution. The following collageno- 

 l)tic properties of tadpole tissues were recently described by the 

 authors (Gross and Lapiere, 1962). 



1. The area of lysis expanded in exponential fashion and was 

 linearly related to the size of the tissue explant. 



2. Living tissues were required for collagenolytic action. Freezing 

 and thawing the explant prevented collagenolytic activity. 



3. The pH of the medium remained in the physiologic range, 

 and up to 80 per cent of the collagen substrate could be digested to 

 dialyzable peptides. No free hydroxvproline was released. 



4. Twelve different tadpole organs were examined for collageno- 

 lytic activity. Only four were active, tail fin and back skin, gill, and 

 gut. These four tissues undergo the most dramatic remodeling and 

 resorption of connective tissue during metamorphosis. The back 

 skin showed considerably less activity than did the tail fin. 



5. Tissue cultures could be used for quantitative microassay; the 

 area of lysis was directly proportional to the release of radioactive 

 collagen fragments when radioactive collagen substrates were used. 

 The lytic process could be readily compared with that of a standard 

 amount of purified bacterial collagenase placed on the gel. 



Further studies have revealed changes in collagenolytic activity 

 in the cultured tail fin tissues of animals in metamorphosis. In the 

 first experiment, groups of tadpoles were exposed to thyroxin for 

 2 and 6 days. Sections of tail fin tissue approximateh' 1 mm- in area 

 were cultured on radioactive collagen gels at 27°C. At appropriate 

 intervals the cultures were assayed for collagenolytic activity. The 

 results are shown in Fig. 12. Two davs of treatment produced little 

 increase in activity. After 6 days, however, when the tail had dimin- 

 ished by approximately 30 per cent in length, collagenolytic activity 



