TABLE II 

 Average Content and Ratios of Pyridine Nucleotides in S. purpuratus^ 



a 10-1° moles/ 105 cells 



TPtm 



TPNH 

 TPN 



DPN 



DPNH 



Unfertilized 



6.7 + 1.5 

 7.0 + 0.1 



59.3 + 6.8 

 3.3 + 2.5 



0.96 



DPN 



17.9 



Total 



7. DPN & DPNH 



7. TPN & TPNH 



76.3 



827. 

 187. 



78.1 



487. 

 527. 



of the TPN? You've got 10^ molecules per cell 

 formed in about 10 seconds. Isn't that quite 

 rapid formation? Is there any "miracle" here? 



EPEL: Any "miracle"? 



POLLARD: I'm referring to the fact that 

 10^ molecules per cell are made in 10 seconds. 



EPEL: The data in the figure is per 100,000 

 cells. 



POLLARD: It's 10^ molecules per cell, 

 which gives a really very rapid turnover num- 

 ber of about 10,000 per minute. Why are the 

 enzymes that good? That would seem to me to 

 be the exciting thing you've got here. Is it all 

 right? 



EPEL: Actually this is consistent with 

 maximum activity of the enzyme. For some 

 reason the enzyme is suddenly activated close 

 to maximum activity, or at least within a factor 

 of 2 or 4. 



POLLARD: That is a slight miracle. Is it 

 more than "maximum"? 



EPEL: No, it's not more than maximum, 

 as extrapolated from in vitro experiments under 

 simulated in vivo conditions. 



DEERING: This assumes you know how 

 much of the enzyme is present. 



EPEL: Yes. On the basis of extracting 

 enzyme from a known amount of cells, and as- 

 saying kinase activity at ATP and DPN concen- 

 trations present in vivo. In any case, if it were 



grossly aberrant, we would notice it. This is 

 the most active source of the enzyme that's 

 ever been found. The maximum activity is only 

 three times less than the 75-fold purified 

 enzyme from pigeon liver. 



B. Respiratory changes 



Simultaneous measurement of respiration 

 rate and cell fluorescence, shown in Fig. 5, 

 indicates that the fluorescence change (TPNH 

 increase) precedes the activation of respiration. 

 Respiration is measured polarographically, and 

 an upward deflection indicates a decrease in 

 oxygen content. Rate is indicated by the slope. 

 The respiration rate (see Fig. 7) is characterized 

 by a transiently large burst, followed by a slow 

 decrease to a rate 4-5 times that of the pre- 

 fertilization rate. Significance of these kinetics, 

 as well as possible controlling mechanisms for 

 respiration, will be described later. 



C. Excretion of the fertilization acid 



Simultaneous measurements of fluorescence 

 and extracellular pH indicate that changes in 

 these two parameters began simultaneously if 

 measured at similar amplification levels (i.e., 

 at amplifications such that the total changes 

 are of similar magnitude on the chart paper), 



21 



