DORMANCY OF BACTERIAL ENDOSPORE 87 



pyruvate and NH3 is clearly established. If the deamination of 

 exogenous alanine is an initial step in germination, then the 

 preferential metabolism of exogenous alanine would be expected 

 in the early stages of germination. This was confirmed by 

 observing the liberation of I'^CO-z from Ci labeled alanine^^. The 

 highest specific activity was observed initially (after only 10 min 

 incubation — the earliest point at wich sufficient CO2 is produced 

 to permit isolation). The findings are at least consistent with the 

 view that the deamination of exogenous L-alanine precedes that 

 of endogenous alanine and may be an initial step. 



NATURE OF THE INITIAL BINDING SITE FOR L-ALANINE 



The above findings suggest that the L-alanine binding site is 

 the deaminating enzyme or an earlier step. If it is the enzyme 

 itself, the specificity of the alanine binding site on the spore 

 should be identical to that of the deaminating enzyme. To 

 provide information for this comparison we have purified the 

 relevant enzyme over 60 fold from activated spores and charac- 

 terized it^^. The purified enzyme, an L-alanine dehydrogenase, 

 carries out a DPN-linked deamination of L-alanine to pyruvate 

 and ammonia. The reaction is reversible, specific for DPN, and 

 has an activation energy of 8,200 cal/mole. The affinity constants 

 for substrates and products as well as the other collective data 

 on its properties identify the enzyme as the same as that reported 

 in Bacillus subtilis vegetative cells by Pierard and Wiame^^, in 

 B. cereiis vegetative cells by ourselves^^ and in Mycobacterium 

 by Goldman^^. 



The amination reaction requires a proton which accounts for 

 the fact that the reverse reaction is favored by more acid 

 conditions. The pH optimum for deamination is high, about 

 pH 10, which is in agreement with the ability of spores to ger- 

 minate at high pH. 



If the L-alanine dehydrogenase is the initial step in germina- 

 tion then it should be (a) inhibited by D-alanine and (b) be active 

 on L-alanine analogs which act as germinating agents. In Table II 



References p. 94 



