scattering (structural changes) and 366 m^ 

 induced cell fluorescence can be made. This 

 latter measurement, in all systems so far 

 described, is specific for detecting changes in 

 reduced pyridine nucleotide (4). Through the top 

 of the cuvette can be inserted an oxygen electrode 

 for measuring respiration rate, and a pH 

 electrode for measuring excretion of the fer- 

 tilization acid (see 6 and 12 for experimental 

 details). Finally, samples can be taken from 

 the cuvette for analysis of coenzymes, sub- 

 strates, or enzyme activity. The four para- 

 meters (light-scattering, fluorescence, respira- 

 tion, and acid excretion) have been monitored 

 through low time constant amplifiers, and re- 

 corded individually on synchronized recorders, 

 or simultaneously on a multi- channel recorder. 



RESULTS 

 I. Temporal sequence of fertilization changes 

 A. Pyridine nucleotide changes 



TPNH is the coenzyme generally involved 

 in reductive biosynthesis, as indicated by the 

 coenzyme specificity of reductive reactions, 

 as well as by the general correlation between 

 synthetic activity and both TPNH levels and 

 TPNH/TPN ratios (7, 8). This compound has 

 been reported to increase within one hour after 

 fertilization (9), and hence this change might 

 be important in initiating and controlling re- 

 ductive biosynthesis in the egg. 



As indicated, 366 m/;f-induced cell fluor- 

 escence is a sensitive monitor of reduced 

 pyridine nucleotide in vivo. Measurements of 

 cell-fluorescence following fertilization, shown 

 in Fig. 1, indicate an increase in this para- 

 meter, beginning at 40 seconds after sperm 



addition, and ending by 5 minutes with a 1/2 

 time of 35 seconds. Enzymatic analyses of 

 reduced pyridine nucleotides in alkaline- 

 extracted cell homogenates are shown in Fig. 2. 

 These indicate that the reduced pyridine nu- 

 cleotide which increases is TPNH, and that this 

 increase parallels the changes in fluorescence. 

 Furthermore, the sum of reduced pyridine 

 nucleotides at various times after fertilization is 

 linearly related to the cell fluorescence (Fig. 3), 

 which confirms the relationship between in vivo 

 fluorescence and reduced pyridine nucleotide. 

 The increase in TPNH does not result 

 f'-om reduction of pre-existing TPN, but rather 

 from phosphorylation of DPN to TPN, and most 

 probably the subsequent reduction of this TPN 

 to TPNH. This is shown in Fig. 4 and Table II. 

 Figure 4 shows that DPN decreases, while TPN 

 increases in a mirror-image fashion. Similar 

 behavior is also seen for the TPNH increase 

 shown in Fig. 2. These changes suggest a 

 precursor -product relationship, and this sup- 

 position is further verifiedby the stoichiometric 

 relationship shown in Table II, which is a 

 balance sheet of pyridine nucleotide before and 

 after fertilization. The pertinent point to observe 

 is that total amount of pyridine nucleotide is the 

 same before and after fertilization, but that an 

 interconversion of pyridine nucleotide types has 

 occurred - total TPN and TPNH increasing, 

 while total DPN andDPNH decrease. The enzyme 

 implicated in such an interconversion is DPN 

 kinase, which catalyzes the reaction: 



DPN and ATP ► TPN and ADP(10, 11). 



This enzyme, then, is apparently activated by 

 fertilization. Possible mechanisms of its acti- 

 vation will be described later. 



POLLARD: How does that fit with any 

 reasonable turnover numbers for the production 



1000 300 



270 



240 



210 



ISO 160 



Seconds 



120 



90 



60 



30 



-30 



Fig. 1. 



366 mu Induced fluorescence of eggs of S. purpuratus following fertilization. (Fig. 1, Epel, 

 Biochem. Riophys. Res. Comm. 17, 69, 1964; reproduced with permission of Academic Press.) 



19 



