L.VARIEGATUS 



S. PURPURATUS 



40 80 120 160 40 60 120 160 



Seconds After Sperm Addition Seconds After Sperm Addition 



Fig. 13. 



Comparison of clianges in content of glucose-6-phosptiate,DPN,TPN and rates of respira- 

 tion in eggs of L.variegatus and S. purpuratus. (From Epel and Iverson, In "Control of 

 Energy Metabolism," 1965; reproduced with permission of Academic Press.) 



EPEL: We don't know yet, but it is another 

 possibility. We're just looking into this now. 



C. TPNH changes 



The stoichiometry and kinetics of the pyri- 

 dine nucleotide changes implicate activation of 

 DPN kinase by fertilization. That this is the 

 case is seen in Table III, which shows activity 

 measurements of DPN kinase in homogenates 

 prepared from unfertilized and fertilized eggs. 

 As can be seen, the activity is essentially the 

 same in both cases. Although this demonstrates 

 that the enzyme is indeed present in the un- 

 fertilized egg, and hence activated by fertiliza- 

 tion, it is disappointing from a heuristic view- 

 point that these differences could not also be 

 reflected in the broken cell preparations. This 

 suggests that the enzyme is either activated 

 by the homogenization procedures, that the 

 enzyme is activated by the assay procedure, or 

 that some substrate, activator, or cofactor 

 missing in the unfertilized egg is being either 

 released during homogenization or supplied in 

 the assay mixture. 



The structural changes, as well as several 

 reports of enzyme translocation following fer- 

 tilization (32, 33), suggested that the enzyme 

 might be changing its subcellular site upon fer- 

 tilization. To check this, the enzyme has been 

 extracted with numerous different media, and 

 the activity in particulate and soluble phases 

 checked. In all cases, the enzyme has always 

 been found in the supernatant. 



Measurement of substrate localization be- 

 fore and after fertilization were also carried 

 out, estimating the amounts of DPN and ATP 

 in the mitochondrial-nuclear fraction and the 

 post-mitochondrial supernatant. Although not 

 completely satisfying from the viewpoint of 

 both leakage of substrates from particles, and 

 some loss of ATP and DPN during centrifugation, 

 the results did not indicate any large amount 

 of binding of ATP or DPN to or in particles. 

 Briefly, 75% of the DPN and greater than 90% 

 of the ATP were in the post-mitochondrial 

 supernatant. As the DPN kinase is also in the 

 supernatant, it appears that both substrates and 

 enzyme are present in adequate amounts for the 

 reaction to proceed. These findings, therefore. 



28 



