are always associated with development, one 

 might well inquire into the possible advantage 

 this situation could bring to the differentiating 

 cell. I would like to suggest that the least pre- 

 carious approach for a differentiating cell 

 actually may reside in its dependence upon a 

 complex interplay of many limiting factors. In 

 this way, unusual deficiencies or abundances in 

 the cell or the cell's environment need not 

 necessarily upset the process of differentiation. 



Let me elaborate on this concept briefly, 

 using some recent work with hexokinase( Fig. 7). 

 In the figure on the left the reciprocal of the 

 glucose concentration is expressed on the 

 abscissa and the inverse of the velocity of the 

 reaction on the ordinate. Velocity is seen to 

 increase with increasing levels of ATP from 0.1 

 to 1.2 millimolar. In other words, the reaction 

 is stimulated by ATP in the presence of limiting 

 levels of glucose. Similarly in the right part of 

 this figure increasing levels of glucose stimulate 

 the reaction in the presence of limiting levels 

 of ATP. When both substrates are limiting, in- 

 creasing the concentration of either increases 

 the rate of the reaction (6). Thus, if this situation 

 prevailed in the cell, increasing levels of either 

 ATP or glucose could increase the level of 

 glucose-6-phosphate. If, on the other hand, either 

 substrate were in excess, G-6-P accumulation 

 would depend upon the concentration of the other 

 substrate. In this sense the system would be less 

 flexible than if both substrates limited. Such 

 flexibility may be very important to the stability 

 and reproducibility of differentiation. 



Let us now turn from complications at the 

 substrate level to even greater complications at 

 the enzyme level. We have said nothing as yet 

 concerning the enzyme activity during the earlier 

 stages of development. Figure 8 shows one of 

 our earlier experiments in which we compared 

 enzyme activity at various stages of development 

 with the amount of alkali-insoluble material 

 present. The stages of development are amoeba 

 (A), aggregation (agg), preculmination (PC), a 

 combination of culmination and fruit (CF) and 

 fruit (F). Aliquots of cells were harvested at 

 various stages of development and the percent 

 dry weight of the cell wall material determined. 

 This is indicated on the left ordinate. At each 

 stage, also, a particulate enzyme of cell wall or 

 cell membrane preparations was prepared in 

 tris buffer and 10""* M EDTA and was incubated 

 with radioactive UDPG. The alkali-insoluble 

 radioactive product was isolated, counted and 

 related to the dry weight of the sample. The 

 specific enzyme activity was thus determined. 



ATP 



GLUCOSE 



0.04 mM 



['glucose] 



atp + glucose 



Fig. 7. 



(Fig. 3, From Silverstein and Boyer, /. Biol. Chem. 239, 

 3645, 1964; reproduced with permission of the American 

 Society of Biological Chemists, Inc.) 



< 



3 



O 



< 



_i 

 < 



I5-- 



I0-- 



5-- 



-tt- 



. f.. A ...t ,-5r - 



--I000 t 



KlF + Agg) 



X 



liJ 



>- 

 cc 



a 



--500 S 



Q. 

 U 



>- 





 HOURS 



STAGE h 



gg 



PC 



H 1 



CF F 



Fig. 8. 



From Wright, Barbara, E.: Control of Carbohydrate 

 Synthesis in the Slime Mold. In Developmental and Meta- 

 bolic Control Mechanisms and Neoplasia (A Collection of 

 Papers Presented at the Nineteenth Annual Symposium 

 on Fundamental Cancer Research, 1965), p. 304. Bald- 

 more, The WiUlams and Wllkins Company, 1965. 



and is expressed on the right ordinate. Mixed 

 preparations of an enzyme that was active with 

 an inactive preparation gave relatively little 

 inhibition. The data in Fig. 8 exhibit a striking 

 correlation in the activity of an enzyme and the 

 accumulation of the product of the activity of 

 this enzyme. The correlation suggests a causal 



115 



