Activity at the enzyme and substrate level 

 must necessarily be correlated in time with the 

 accumulation of the product characteristic of the 

 differentiated cell. This need not be true, of 

 course, for the nucleic acid template responsible 

 for the presence of these enzymes. In fact, 

 recent studies of Brown in the amphibian. Gross 

 in the sea urchin and Sussman in the slime mold 

 indicate that certain stages of differentiation do 

 not, in fact, depend upon the concurrent forma- 

 tion of messenger RNA. This situation brings 

 renewed interest to other types of control, such 

 as the activation of preformed mRNA, enzyme 

 accumulation through lack of degradation, en- 

 zyme relocation within the cell, the availability 

 of a substrate or an enzyme activator, etc. 

 Regardless of the relative importance of nucleic 

 acid control during a particular process of dif- 

 ferentiation, the cellular environment of the 

 enzymes involved is critical, of course, in de- 

 termining the nature and extent of their activity. 

 In other words, since the action of an enzyme is 

 entirely dependent upon levels of specific sub- 

 strates, activators, inhibitors and the like, 

 knowledge of these variables in the intact cell 

 is essential in attempts to evaluate the signifi- 

 cance either of a constant or a changing enzyme 

 level to a reaction important to development. 



Let me illustrate this point by mentioning 

 just two examples in the slime mold. The mor- 

 phogenesis of this microorganism depends, in 

 part, upon the breakdown of endogenous protein 

 and its eventual conversion to carbohydrate. As 

 protein degradation intensifies during develop- 

 ment, the intracellular concentration of gluta- 

 mate increases an order of magnitude. Oxidation 

 of this amino acid and its entry into the Kreb's 

 cycle is a necessary step in its utilization for 

 carbohydrate synthesis. The enzyme responsible 

 for this oxidation, glutamic dehydrogenase, is 

 very stable in extracts prepared throughout 

 development. Although the concentration of this 

 enzyme does not change, its activity when meas- 

 ured in vivo using radioactive glutamate in- 

 creases 7-fold during development. The dehy- 

 drogenase was purified and its affinity for 

 glutamate was determined; knowing the effect 

 of substrate concentration on the rate of this 

 reaction, it was shown that the accumulation of 

 glutamate in vivo could fully account for the 

 enhanced rate of the reaction in differentiating 

 cells. Thus, data at the enzyme level was insuf- 

 ficient in interpreting the in vivo activity of this 

 enzyme during development (1). 



The slime mold offers another example of 

 an enzyme which does increase in concentration 



during development (some 6-fold), yet this 

 change is not reflected in its activity in vivo. 

 Dr. Gezelius has studied an alkaline phosphatase, 

 highly specific for 5' -AMP, which reaches a 

 maximum concentration at the end of differen- 

 tiation. However, inhibition of the enzyme by 

 increasing levels of inorganic phosphate in vivo 

 results in maximum activity of the enzyme not 

 at the end but in the middle of differentiation (2). 

 Thus, observed alterations in the concentration 

 of an enzyme may not bear a direct relationship 

 to its actual activity in the differentiating cell. 

 This is probably the rule rather than the excep- 

 tion. 



Enzymes are usually measured under con- 

 ditions of pH, inonic strength, substrate con- 

 centration, co-enzyme, activator or inhibitor 

 concentrations, which do not reflect the condi- 

 tions in the differentiating cell. Much more 

 data are needed in which enzyme activities are 

 measured both in vivo and in vitro and in which 

 levels of relevant substrates, co-enzymes and 

 activators are determined in vivo at various 

 stages of development. All these data, taken 

 together, may then give a consistent picture of 

 the activity of an enzyme in differentiating 

 cells. 



To facilitate the following discussion, I 

 will very briefly summarize the life cycle of 

 D. discoideum (Fig. 2). Upon starvation, the 

 cellular slime mold passes from the vegetative 

 stage, during which it exists as a homogeneous 

 population of myxamoebae lacking a cell wall, 

 through an aggregation process to become a 

 differentiated multicellular organism. Succes- 

 sive stages which I will refer to are known as 

 aggregation, pseudoplasmodium, preculmina- 

 tion, culmination and sorocarp or fruiting body. 

 In the terminal stages of development the cells 

 are ensheathed in a cell wall composed of a 

 cellulose-glycogen polysaccharide complex, the 

 synthesis of which will be the subject of a good 

 portion of my presentation. All of the experi- 

 ments I will talk about were done with cells 

 which were starving on 2% agar throughout the 

 differentiation cycle. 



Figure 3 summarizes the general area of 

 metabolism with which we will be concerned. 

 Endogenous material, such as protein, is de- 

 graded and gluconeogenesis begins. Hexose 

 phosphates are formed andglucose-1-phosphate 

 together with UTP unite to form uridine di- 

 phosphoglucose (UDPG), an essential precursor 

 to cell wall material. Phosphoglucomutase, 

 interconverting G-l-P and G-6-P, is very 

 active throughout development, as is pyro- 



110 



