ENZYMES ACTIVE IN THE INTACT SPORE 101 



question: can the apparently dormant deaminase be activated prior to 

 germination or is germination per se the activating step? 



Perhaps one of the most controversial questions encountered in reference 

 to these thermal-resistant enzymes is whether these catalysts are biological. 

 Because they are particulate in nature, one finds it difficult to demonstrate 

 normal protein behavior using methods generally employed for common 

 enzymes. No doubt the strongest evidence in support of the enzymatic nature 

 of these catalysts is that autoclaving completely destroys activity in 15 

 minutes. Also, a general procedure has been described by Stewart and 

 Halvorson (1954) by which a suspension of particulate alanine racemase 

 was separated into a soluble, heat-labile fraction and a particulate, heat- 

 resistant fraction by ultrasonication and subsequent centrifugation. Further 

 evidence has shown that the soluble heat-labile fraction was susceptible to 

 pepsin digestion whereas the particulate fraction was completely stable. Re- 

 sults analogous to that reported for the racemase have been indicated in our 

 laboratory with the nucleoside ribosidase of terminalis (Nakata, 1956). 



The effects of the heavy metals Cr+ + , Cu++, and Hg++ in trace amounts 

 (10~^ M) were also studied and observed to completely inhibit ribosidase 

 and racemase activities, further supporting an enzymatic system (unpublished 

 data). 



The significance of the alanine racemase system in B. terminalis was in- 

 vestigated by Church et al (1954) and by Harrell and Halvorson (1955) 

 who reported it to play no active part in the germination process. The 

 significance of the nucleoside ribosidase and the adenosine deaminase sys- 

 tems, however, to my knowledge, has not as yet been defined. Dr. Lawrence 

 (1955a), w^orking with the ribosidase in whole spores and spore extracts of 

 B. terminalis, has demonstrated the adenosine cleavage to adenine and free 

 ribose. Although the recovery of ribose as compared with the adenosine 

 cleaved was low at 37°, no enzymes were detected in spore extracts which 

 metabolized the liberated ribose. Inosine, the deaminated product of adeno- 

 sine, was not implicated in his studies. 



Dr. Powell, working with germinated spores of B. cereus, also observed 

 low ribose recovery when these spores were incubated with adenosine alone, 

 but noted the disappearance of adenosine with formation of adenine and 

 hypoxanthine (Powell and Hunter, 1956). When these germinated spores 

 were heated 15 minutes at 60° prior to incubation with adenosine, expected 

 ribose values were obtained. The interpretation that ribose was metabolized 

 by the germinated spores was questionable since analysis of the homogenates 

 failed to reveal such enzymes. 



Considering these and the data obtained in our laboratory with fresh 

 B. terminalis spores, a more feasible explanation is suggested. The spores 



