540 Energy Exchange and Enzyme Development During Embryogenesis 



enzyme was the same before fertilization as 

 afterward. Therefore, he concluded that the 

 increased respiration and sensitivity to cya- 

 nide after fertilization were not due to syn- 

 thesis of new enzyme but were brought 

 about by enhancement of the activity of 

 enzyme previously present. In some way, 

 fertilization had accomplished a change in 

 the relationship of the enzyme with other 

 links in the respiratory chain. 



Later work by Krahl et al. ('41) confirmed 

 these observations. Cytochrome oxidase ac- 

 tivity, measured in the presence of cyto- 

 chrome c, was the same in unfertilized and 

 fertilized eggs, and the enzyme seemed to 

 have the properties usually associated with 

 cytochrome oxidase from other sources. It 

 is of interest to note that cytochrome oxidase 

 is not localized in mitochondria but rather 

 is associated with non-mitochondrial par- 

 ticulates in the supernatant solution of 

 centrifuged egg homogenates (Hutchens, Ko- 

 pac, and Krahl, '42).* Gustafson ('52) has 

 assumed that cytochrome oxidase is grad- 

 ually built into the mitochondria at the 

 time when the mitochondrial population 

 increases after the mesenchyme blastula 

 stage (Gustafson and Hasselberg, '51; 

 Deutsch and Gustafson, '52; Gustafson and 

 Lenicque, '52). 



Cytochrome c is abundant in sea urchin 

 sperm (Ball and Meyerhof, '40; Rothschild, 

 '48), but so far it has not been detected in 

 the egg. However, both Ball and Meyerhof 

 and Borei ('50) observed absorption bands of 

 iron porphyrins related to the cytochromes, 

 and Borei believes that sub-detectable 

 amounts of cytochrome may nevertheless be 

 present and functionally significant. 



That sea urchin eggs contain dehydro- 

 genases was first demonstrated by Runn- 

 strom, and this was confirmed by Ballentine 

 ('38, '40), who showed in addition that the 

 total dehydrogenase activity after fertiliza- 

 tion was approximately three times greater 

 than before. However, attempts to discover 

 the various specific dehydrogenases con- 

 cerned with the operation of the citric acid cy- 

 cle were unsuccessful. The absence of succinic 

 dehydrogenase has been frequently reported 

 (Ball and Meyerhof, '40; Ballentine, '40; 

 Krahl et al., '41), but recently Gustafson 



* Weber (personal communication) has obtained 

 results which are at variance with those previously- 

 reported. He has found that approximately 90 

 per cent of the cytochrome oxidase of homogenates 

 of imfertilized eggs of Paracentrotus lividus can be 

 recovered in the particulate fraction sedimenting 

 at 600 to 12,000 X g. 



and Hasselberg ('51) claimed to have found 

 the enzyme in egg homogenates from two 

 species of sea urchin. It should be noted, 

 however, that Bodine, Lu, and West ('52) 

 showed that reduction of triphenyltetrazol- 

 ium chloride, used by Gustafson and Hassel- 

 berg to measure succinic dehydrogenase 

 activity, is not a specific test for the enzyme. 

 DPN was found to be present in Arbacia eggs 

 by Jandorf and Krahl ('42), and Barron and 

 his co-workers were able to show that the 

 eggs covild oxidize pyruvic acid (Goldinger 

 and Barron, '46). This was also shown to 

 be true for the eggs of Echinus esculentus 

 by Cleland and Rothschild ('52b). 



It seems, therefore, that the dehydrogen- 

 ases of the citric acid cycle must be present 

 but that something in crude egg homogen- 

 ates interferes with their activity. A possible 

 explanation of the failure of others to ob- 

 serve active specific dehydrogenases was 

 offered by Keltch et al. ('50) when they 

 showed that echinochrome inactivates a num- 

 ber of dehydrogenases by oxidizing their 

 sulfhydryl groups. Indeed, they foimd that 

 cell-free, non-mitochondrial particulate sys- 

 tems of unfertilized eggs could oxidize 

 a-ketoglutarate, oxaloacetate, and succinate, 

 and they demonstrated that generation of 

 high-energy phosphate bonds accompanied 

 these oxidations. Thus, they confirmed the 

 earlier observation of Lindberg and Ernster 

 ('48) of oxidative phosphorylation by homo- 

 genates of Strongylocentrotus droebachiensis 

 eggs. Clowes, Keltch, Strittmatter, and Wal- 

 ters ('50) went on to show that oxidative 

 phosphorylation could be inhibited by nitro- 

 or halo-phenols, and it was concluded that 

 generation of high-energy phosphate bonds 

 occurs, as it does in muscle or kidney, 

 through the citric acid cycle. 



That the citric acid cycle operates in the 

 egg of Echinus esculentus has been con- 

 vincingly demonstrated by Cleland and 

 Rothschild ('52b). Evidence for the presence 

 of the enzymes in the cycle rests upon (1) 

 demonstration that malonate, long known to 

 act as an inhibitor of siiccinic dehydrogenase, 

 blocks the endogenous respiration of egg 

 homogenates, (2) finding that oxygen con- 

 sumption of egg homogenates can be stimu- 

 lated by all of the intermediates in the citric 

 acid cycle (see Table 21), and (3) demon- 

 stration of the complete oxidation of pyruvic 

 acid. 



Evidence for the operation in sea urchin 

 eggs of a typical Embden-Meyerhof glyco- 

 lytic cycle has also been provided. Gustafson 

 and Hasselberg ('51) demonstrated the pres- 



