ENZYMES ACTIVE IN THE INTACT SPORE 99 



shocking; (2) the addition of activating suhstances or the combination of 

 heat shocking with activating substances; and (3) prolonged storage. 



(1) Prolonged heat shocking. The effect of prolonged heat shocking has 

 been shown by several investigators (Powell and Hunter, 1955; Murty and 

 Halvorson, 1957b; Church and Halvorson, 1956) to alter the physiological 

 behavior of spores without germination. As an example, the germination 

 requirement of B. terminalis has been observed to change where L-alanine 

 no longer is a requisite for the germination of these heat-treated spores. 

 Heat shocking of spore suspensions prior to disintegration has also been 

 shown to have various influences on the activities of enzymes found in the 

 subsequent spore-free extracts. In one of the more recent studies, Dr. Krask 

 (1956) has reported a 2-fold increase in acetokinase-like activity in B. 

 terminalis after 15 minutes of heat shocking prior to disintegration and a 

 16-fold increase after 1 hour. This and other similar reports well indicate, 

 then, that some sort of activation is occurring during prolonged heat shock- 

 ing. These findings, correlated with the evidence that dipicolinic acid is 

 gradually released during heat shocking (Harrell, 1956) may have direct 

 bearing on the activation of some enzymatic systems in intact spores. 



^2) Addition of activating substances. Oftentimes enzymatic activation 

 occurs unnoticed to the investigator since small amounts of a particular sub- 

 stance may be all that is necessary for the activation of some systems. This 

 has been well demonstrated by the glucose oxidizing system in the spores 

 of B. terminalis, where trace quantities of L-alanine together with prolonged 

 heat shocking were responsible for the activation of an enzyme system previ- 

 ously dormant or undetectable in the untreated spores. This will be discussed 

 in greater detail by Dr. Murty and Dr. Church in the following papers. 



(C) Thirdly, a few of the past investigations have revealed enzymes 

 in spore homogenates and spore-free extracts prepared from dormant spores, 

 and these enzymes, although undetected in the dormant intact spores, were 

 assumed to be actively present prior to disintegration. Unless the enzymatic 

 activity can be demonstrated with intact spores under conditions satisfying 

 the suggested criterion, or one similar to it, the validity of these assumptions 

 is open to criticism. Enzymes that may be active in spore homogenates and 

 extracts are not necessarily active in the dormant whole spore. During ultra- 

 sonic or mechanical disruption a variety of substances are released from 

 the spores which conceivably can result in the following: (1) the germination 

 of unbroken spores in the homogenates, whose subsequent activities may be 

 erroneously attributed to the ungerminated spores, or (2) the activation of 

 enzymes previously undetectable in the intact spores but highly active in 

 the subsequent extracts as a result of the disintegration process. Viable counts 

 on the extracts are strongly recommended to rule out the first possibility. 



