relationship, but we shall see that this is not 

 justified. We found that if one goes from lO-^M 

 EDTA to 0.1 MEDTA, it is possible to partially 

 stabilize enzyme at the earlier stages of develop- 

 ment and detect activity. 



Table VIII is a stage study in which we 

 harvested, killed the cells and isolated enzyme 

 at two different stages, late aggregation and 

 culmination, in the presence of 0.01 M EDTA 

 and 0.1 M EDTA. In other words, we had four 

 enzyme preparations: two at aggregation, two 

 at culmination. The enzyme preparations were 

 made and assayed as quickly as possible; then, 

 they were stored in an ice bath at 5° and were 

 assayed again at two hours and at 24 hours. As 

 you can see, at both concentrations of EDTA 

 the enzyme activity at aggregation decreased 

 with time, but was spared to a greater extent 

 at O.l M EDTA. At culmination, however, the 

 enzyme activity was not only spared but in- 

 creased. This enzyme is strikingly activated 

 by high concentrations of EDTA. Clearly in- 

 activation of the enzyme prepared at aggrega- 

 tion was more rapid between hour and hour 2 

 than from hour 2 to hour 24. In other words, 

 the inactivation curve drops rapidly at first and 

 then more gradually. Seeing this, one wonders 

 what happens between the time that the en- 

 zyme was first prepared and the time that the 

 hour value was obtained. In other words, in 

 that period of preparing the enzyme inactiva- 



tion may have been even more rapid. At any 

 rate, enzyme prepared at one stage is more in- 

 activatable than the enzyme prepared at a later 

 stage. 



We call this phenomenon differential in- 

 activation. It is an in vitro artifact, fairly com- 

 mon in the slime mold. We are very impressed 

 with the extreme difficulty of detecting it, since 

 it took us almost a year in this case. In each 

 stage study the period in time and in stage at 

 which the enzyme activity can first be detected 

 and shown to be unstable varies and is very 

 short-lived. Until this enzyme is stabilized, we 

 cannot determine specific enzyme activity as 

 a function of developmental stage. Our experi- 

 ence with differential enzyme inactivation makes 

 us very suspect of the absence of any enzyme 

 activity and prone to place faith in changes in 

 enzyme activity only when (1) the enzyme is 

 detected and (2) is relatively stable or capable 

 of being stabilized. It is possible that the cell 

 wall enzyme under study is always present in 

 the cell membrane but is undetectable in vitro 

 due to the absence of stabilizing primer, for 

 example. We have some preliminary data on this 

 point, but before presenting it, I would like to 

 summarize the facts briefly (Table IX). 



We have recently found 4 enzyme activities 

 involved in cell wall or glycogen synthesis or 

 both (see Table IX). They may be the same 

 enzyme or some of them may be different, at 



TABLE VIII 

 Effect of EDTA Concentxatlon on Initial Enzyme Activity and Stability^ 



0.35 mg dry weight 

 "0.50 mg dry weight 



* From Wright, Barbara E.: Control of Carbohydrate Synthesis in the Slime Mold. In 



Developmental and Metabolic Control Mechanisms and Neoplasia (A CoUectlon of Papers Pre- 

 sented at the Nineteenth Annual Symposium on Fundamental Cancer Research, 1965), 

 p. 308. Baltimore, The Williams and Wilkins Company, 1965. 



116 



