110 G. G. KRISHNA MURTY 



Discussion. The results obtained by Murrell, Church and Halvorson and 

 us lead to some common conclusions. It is evident that the oxidative 

 enzymes normally dormant in the intact spores can be activated in the 

 absence of any detectable germination. 



The mode of activation may vary with the age and history of the spores. 

 As has already been pointed out, we were able to detect small amounts of 

 alanine in the washings from aged spores and this probably explains the 

 activation of these enzymes by heat alone as noted by Church and Halvorson 

 and by us. So it is safe to conclude that L-alanine is responsible for the 

 activation of the enzymes. 



Once the spores have been activated, inhibitors have no effect, although 

 the activation seems to decay with time. The rate of oxidation of glucose 

 is a function of the number of spores and the concentration of L-alanine, the 

 concentration of L-alanine being limiting in the case of dense suspensions. 



However,, there are some significant differences also. While in the case 

 of spores of B. suhtilis the presence of L-alanine is necessary for the maximum 

 rate of oxidation of glucose, in the case of B. terminalis spores the L-alanine 

 can be washed out after heat activation without any loss of activity. 



The decrease in the capacity of B. suhtilis spores pretreated with alanine 

 to oxidize glucose and L-alanine is similar to the loss in glucose-oxidizing 

 activity noted on washing germinated spores of B. terminalis. While in the 

 case of germinated B. terminalis spores, the loss of activity on washing may 

 be due to washing out part of the cofactors involved, the situation with the 

 spores of B. suhtilis is considerably more complicated. 



Murrell has noted a decrease in activity with increasing time of incubation 

 of spores in L-alanine alone before adding glucose as the second substrate. 

 Church and Halvorson have noted that the restoration of activity of B. 

 terminalis spores on second heat shock fell off with the time elapsed after the 

 first heat activation. In the case of dense spore suspensions we found that 

 alanine had to be added before the second heat shock for the enzymes to be 

 activated, suggesting the possibility of alanine being metabolized during the 

 activation or oxidation of glucose. All these observations can probably be 

 explained on the simple assumption that the activated spores were also 

 metabolizing L-alanine. This would suggest that we should look more care- 

 fully for an enzyme metabolizing alanine in the activated spores. As Mr. 

 Nakata has pointed out earlier, the adenosine deaminase may be another 

 enzyme one should look for in activated spores. 



The release of alanine during storage suggests the possibility of proteolytic 

 activity, and with the proper techniques of activation one may be able to 

 demonstrate the presence of proteolytic enzymes in the intact spores as pro- 

 teolytic enzymes have been demonstrated in spore-free extracts. 



