ENZYMES ACTIVE IN THE INTACT SPORE 95 



lack the enzyme, require L-alanine for germination. At a pH of 11.3, the 

 enzyme is inactive, but B. terminalis spores would still germinate in the 

 presence of alanine and adenosine. At present, it is not known what role, 

 if any, this enzyme plays in the economy of the spore. I believe Dr. Church 

 has evidence that alanine is deaminated to yield pyruvic acid and ammonia 

 in the presence of Avhole spores or spore extracts. I would also like to ask 

 about the peak activity of the racemase which was reported to occur in young 

 vegetative cells, about 4 hours old. 



Catalase. In a number of cases, spores have been washed until they 

 exhibit no catalase activity. At this point, they have been considered clean. 

 With B. terminalis spores such a point was not reached. Even after pro- 

 longed washing, the level of activity remained constant, suggesting that the 

 enzyme was an integral part of the cell (Lawrence and Halvorson, 1954). 

 It was found that the enzyme activity of the intact spore was completely 

 stable for 10-20 minutes at 80 °C, while vegetative catalase was destroyed by 

 3 minutes at this temperature. 0.01 molar concentrations of KF, NaNs 

 or NaCN produced 50-60% inhibition, and 0.02M Na ethylenediamineletra- 

 acetate had no effect. It was found that by disrupting the spores the specific 

 activity increased, and a portion of this activity was then sensitive to 5 

 minutes at 80 °C. As Foster pointed out (1956) it is not known whether the 

 catalase activities of spores and of vegetative cells are caused by identical 

 enzymes. W. G. Murrell, in his Ph.D. thesis at the University of Oxford 

 (1952) also found a heat-stable catalase in intact spores of B. subtilis. 



Adenosine deaminase. Powell and Hunter (1956) noted that for their 

 strains of B. cereus and B. anthracis inosine was a more effective germination 

 stimulant than was adenosine. This suggested to them that inosine might be, 

 in fact, the active compound, and that adenosine might be a precursor of 

 inosine for these spores. The presence and activity of the deaminase was 

 verified by noting the change in UV absorption (peak shifts from 260 to 250 

 millimicrons) and by measuring the ammonia liberated. No oxidation nor 

 transamination was detected. The enzyme in the intact spore was heat 

 resistant, surviving several hours at 60 °C, but in extracts and homogenates 

 the activity was destroyed in 15 minutes at this temperature. Cytidine was 

 attacked at nearly the rate of adenosine, but in spore extracts, adenine, 

 guanine, guanosine and cytosine were not deaminated. It is of interest to 

 note that there was no correlation between the rate of deamination and the 

 rate of germination in adenosine. Neither a preheating of the spores at 

 60°C for 2 hours, nor the addition of L-alanine plus L-tyrosine had any 

 effect on the rate of deamination. although the germination in adenosine 

 alone was remarkably stimulated by 1-hour heat treatment. These workers 

 found that the enzyme was also present in germinated cells in a heat sensitive 



