TABLE ni 

 DPN Kinase Activity in Homogenates of S. purpuratus 



Unfertilized 

 Fertilized 



3.1 + 0.1 



3.2 + 0.04 



Eggs homogenized in 0.1 M trlethanolamine buffer. 01 ml 

 of this extract (1.1 to 1.3 mgms protein) was incubated 

 for 30 minutes at 30°C in a medium containing 5 //moles 

 ATP, 5 |/moles DPN, 20 //moles MgCl2 and 180 //moles 

 trlethanolamine buffer, pH 7.4, in a total volume of 2.0 

 ml. Assay procedures as In Fig. 14. 



suggest that enzyme and substrate are "ap- 

 parently available" to each other, but do not 

 interact until after fertilization. 



The remaining requirement for DPN kinase 

 enzyme activity is a divalent cation. Although 

 we as yet have no data on cation content of 

 the soluble phase, Mazia (34) has shown that 

 fertilization results in an increase in free 

 Ca+2 (as opposed to bound, or non-dialyzable 

 Ca"'"^). Could the enzyme requireCa'''^, andif so, 

 could the Ca+2 change account for enzyme acti- 

 vation? 



Studies to test this hypothesis have been 

 done by assaying enzyme activity in dialyzed 

 or chromatographically desalted supernatants 

 at ATP and DPN concentrations paralleling the 

 in vivo concentrations of substrate. One such 

 activity curve is shown in Fig. 14. It is seen 

 that the enzyme exhibits a requirement for a 

 divalent cation, and is activated more strongly 

 by Ca^^ at low cation concentrations. Above 

 3mM, however, it is seen that Mg'''^ activates 

 20% better than Ca"*"^. Such behavior is rela- 

 tively unique for a kinase, since most enzymes 

 of this type are better activated by Mg''"2, and 

 in some cases are Ca+2 inhibited. For example, 

 Ca+2 is only 40% as active as Mg+2 in pigeon 

 liver DPN kinase (11). 



What picture emerges from these studies? 

 The kinetic analysis suggests the following 

 picture. A light-scattering change occurs, prob- 

 ably reflecting the breakdown of cortical gran- 

 ules. Coincident with this is the initiation of 

 fertilization acid excretion, probably reflecting 

 the release of sulfated mucopolysaccharides. 

 Within a second or two of these two changes 

 DPN kinase is activated. Shortly thereafter (or 

 simultaneously) carbohydrate flux increases, 

 possibly through phosphorylase activation, and 

 when sufficient substrate has reached the res- 

 piratory chain, respiratory activation occurs. 



1 1 1 \ \ I I I r 



10 20 30 40 50 60 70 80 90 100 

 CATION CONCENTRATION (10'"* M) 



Fig. 14. 



Cation dependence of DPN kinase from unfertilized eggs 

 of 5. purpuratus. 0.1 ml of a 12,500g supernatant, desalted 

 by passage through a Bio-Gel P-2 column, was incubated 

 with 0.2 mM DPN, 3.6 mM ATP and the noted concentra- 

 tion of cation In trlethanolamine buffer, pH 7.4, 0.083 M 

 for 30 minutes at 30°C. The reaction was quenched by 

 boiling, and TPN assayed with isocitrlc dehydrogenase. 



The above hypothetical scheme is compatible 

 with the data. The weakest point is the picture 

 of respiratory activation, since there is not 

 really good evidence that carbohydrate mobil- 

 ization via phosphorylase is the responsible 

 factor. 



Although the mechanisms for these changes 

 are not rigorously defined, the analysis of the 

 DPN kinase reaction suggests the hypothesis 

 that the change in free Ca+2 is the primary 

 activator of this enzyme. Glycogen phosphoryl- 

 ase can also be Ca+2 activated through the 

 complex phosphorylase kinase system (35) so 

 such a hypothesis takes on added interest. A 

 possible criticism of this interesting theory is 

 that the kinase is also Mg+2 activated, and the 

 Mg+2 content in vivo is more than adequate to 

 activate the enzyme (36). Although no data is 

 available on the free Mg+2 content, it should be 

 noted that the amount of RNA in the eggs is 

 sufficient to completely bind the available Mg+ 2 

 (37). Obviously much more data has to be ob- 

 tained in order to prove or negate the Ca+2- 

 activation hypothesis, and it is presented here 

 solely to indicate the possible directions of 

 this research. 



In closing, I should like to comment on 

 how the various post-fertilization reactions 

 might be involved in initiating the syntheses 

 characteristic of development. Monroy et al. 

 (3) have recently reported evidence indicating 



29 



