124 



HILLEL S. LEVINSON 



REACTION 

 MIXTURE 



DAYS 



0Q^ 



^ O 



o O O 



o o O 



QOQ 



o o o 

 o o o 



O o o 



- O Q Q Q O 



o o o o 







Fig. 3. Tracing of paper partition chromatogram showing hydrolysis of 

 gelatin by spore extract. G =: gelatin (1.0 per cent) ; X = spore extract 

 (equivalent to 2.7 mg ground spores per ml reaction system) ; AX ^ auto- 

 claved spore extract; Mn := manganese (10 ppm ) as manganous sulfate. 

 Mixtures chromatogrammed after 1, 2, and 3 days of incubation at 30°C 

 (Levinson and Sevag, 1954b). 



to activate protease or peptidase of the spores, which acting on spore ma- 

 terial can produce amino acids or simple peptides which in turn stimulate 

 the spore to germination. The site of action of these amino acids is another 

 related problem which I am not prepared to discuss just now, but perhaps 

 the L-alanine acts as Dr. Murty (this Symposium) has indicated. 



We have some evidence that the spores of B. megaterium do contain pro- 

 teolytic enzymes (Levinson and Sevag, 1954b). Using 1.0 per cent solutions 

 of dialyzed gelatin and egg albumin as substrates, we have demonstrated the 

 presence of proteolytic enzymes in spore extracts by several methods. 



(1) Paper partition chromatography for the detection of amino acids. 

 With gelatin as the substrate ( Fig. 3 ) , amino acids resulting from protein 

 hydrolysis were observable after incubation of the protein with spore ex- 

 tract. The colors were much more intense when manganese was used, and 

 in addition, the liberation of the amino acids from gelatin was accelerated 

 with manganese. Much the same general picture is seen with egg albumin 

 as the substrate (Fig. 4). Without manganese, there was no hydrolysis in 



