670 C. M. LAPIERE AND J. GROSS 



after 6 da) s of continuous exposure to the liormone. Groups of ani- 

 mals were sacrificed at chosen inter\als up to 30 hours; the tail fins 

 and back skins were removed, minced, and extracted three times 

 with 5 volumes of cold 1 m NaCl, followed bv three extractions 

 with cold 0.1 M acetic acid. The extracts were dialvzed against 

 large volumes of water, quantitatively precipitating the collagen 

 with a small amount of globulin, leaving other noncoUagenous pro- 

 tein in tlie supernatant fluid. The dialyzate contained most of the 

 free proline and was dried by lyophilization. The material inside 

 the bag was separated by centrifugation and the precipitates and 

 supernatants were hydrolyzed. Hydroxyproline and proline were 

 isolated from paper chromatograms developed with a phenol-water- 

 ethanol solvent (Tanzer and Gross, in press). Hydroxyproline 

 and proline are widely separated on such chromatograms and may 

 be obtained from the paper in isotopically pure form. An aliquot 

 part of the eluted imino acids was chemically measured and another 

 portion counted in a liquid scintillation counter.* 



It is important to note that although the labeled proline was 

 always given in a single injection, these are not truly "pulse" ex- 

 periments, since, as seen from Fig. 4, the specific activity of free 

 proline levels off rapidly at a fairly high level, approximately 10,000 

 cpm/i"^niole even at 30 hours post injection. 



Metabolism of Total Collagen 



Let us first examine the incorporation of proline-H'^ into the 

 whole collagen pool (measured as hydroxyproline) of back skin 

 and tail fin. The data represent the sum of all three fractions. 



Because of the diminishing tail size and change in water content, 

 net incorporation of label could not be satisfactorily related to a 

 per gram wet weight basis. As the fins and back skins were removed 

 in a standard manner, reproducibly obtaining most of the tissue, 

 the above measurements could best be expressed on the basis of 

 content per "organ." 



Over the 30-hour period of measurement there was only an 11 

 per cent loss of collagen from the tail fin and 9 per cent from the 

 back skin. The net loss of collagen from back skin may only be ap- 



* All these procedures will be described in detail in the definitive publications. 



