544 Energy Exchange and Enzyme Development During Embryogenesis 



the degree of enzyme inactivation. As will 

 be recalled, Gustafson has claimed that 

 cytochrome oxidase is gradually taken up 

 into mitochondria at the time when they 

 increase in the mesenchyme blastula stage. 

 These results may indicate, therefore, that 

 the sensitivity of the enzyme to freezing 

 and thawing is greater after incorporation 

 into mitochondria than before. In the frog 

 embryo, Spiegelman and Steinbach ('45) 

 failed to find any increase in cytochrome 

 oxidase between stages 6 and 19 (Shumway), 

 although respiration of intact embryos rose 

 approximately 800 per cent during this 



Fig. 203. Developmental changes in total nitro- 

 gen (N), volume (V), respiration (/?), and succin- 

 oxidase (S) in the central nervous system of Am- 

 blystoma punctatum. Only the dimensions of the log 

 scale are shown on the ordinate. Initial logarithmic 

 values for 7Vzrl.06; for F=4.81; for R—2.5Q; and 

 for 5=2.50. Abscissa denotes hours of development 

 from fertilization. Volimie data from Boell ('48); 

 data for nitrogen, respiration, and succinoxidase 

 from Boell and Shen (impublished). 



period. They have interpreted this result 

 as showing that what changes during de- 

 velopment is not the amount of enzyme 

 but the spatial orientation of enzyme and 

 substrate. It will be recalled that the same 

 idea had been expressed earlier for the 

 sea urchin egg by Runnstrom ('30). The 

 conclusion that a change occurs during de- 

 velopment in the relationship of enzyme 

 with its normal substrate is reasonable, 

 but its validity would not be impaired by 

 the demonstration that enzyme synthesis 

 occiurs. Brachet ('49) has pointed out that 

 determination of cytochrome oxidase ac- 

 tivity is difficult in frog embryos owing to 

 the large endogenous oxygen uptake of egg 

 homogenates, but by means of a spectro- 

 photometric method he has shown a synthesis 

 of cytochrome oxidase similar to that ob- 

 served in Amblystoma. 



In the grasshopper, salamander, and chick 

 embryos, cjrtochrome oxidase increases rela- 

 tively more rapidly than respiration. It is 



clear, therefore, that although most of the 

 respiratory exchange may proceed through 

 cytochrome oxidase, the concentration of 

 enzyme is not the factor that limits the 

 rate of respiration or of respiratory increase. 

 A number of suggestions have been made as 

 to the cause for limitation of respiratory 

 rate, such as the amount of substrate avail- 

 able or the rate at which it is mobilized, 

 affinity between enzyme and substrate, or 

 limiting concentrations of essential inter- 

 mediates such as cytochrome c or coenzyme. 

 All of these factors are of importance, no 

 doubt, in influencing the rate of respiration, 

 but it would seem that the chief factor 

 responsible for the respiratory rate at a par- 

 ticular time in development is the energy 

 requirement of the embryo. 



Changes in succinoxidase activity have 

 been determined in the salamander (Boell, 

 '48) and the chick (Albaum et al., '46). 

 In Amblystoma, the rate of increase during 

 development is probably not significantly 

 different from that of cytochrome oxidase. 

 Shen ('49) has obtained similar results 

 with mitochondrial preparations from de- 

 veloping rat muscle. Cytochrome oxidase 

 and succinoxidase increase exponentially be- 

 tween the fifteenth day of gestation and the 

 second week after birth, and the slopes ot 

 the developmental curves are identical. In 

 the chick, however, the two enzymes in- 

 crease at somewhat different rates between 

 the second and sixth days of development. 



It has been suggested that enzymes con- 

 cerned with general metabolic processes, 

 such as respiration, develop in relation to 

 the growth of the embryo (Boell, '48). In 

 Amblystoma, the developmental curves for 

 respiration, cytochrome oxidase, succinoxi- 

 dase, and alkaline phosphatase (Krugelis, 

 Nicholis, and Vosgian, '52), as measured 

 in homogenates of the entire embryo, have 

 fairly similar slopes, and the curves seem 

 best interpreted as reflecting the synthesis 

 of metabolically active material from yolk 

 reserves. The relationship between enzyme 

 sjmthesis and growth in the central nervous 

 system (brain plus spinal cord to the level 

 of the sixth somite) of Amblystoma is shown 

 in Figure 203. Here it can be seen that 

 respiration and succinoxidase increase ap- 

 proximately in parallel with size of the 

 nervous system, as determined by total ni- 

 trogen or volume. In the chick embryo, 

 where the relation between enzyme synthesis 

 and growth can also be tested directly, it 

 appears that increase in cytochrome oxidase 

 is proportional to increase in total nitrogen 



