10 60 35 55 75 



% GENERATION TIME % 36 HR. GROWTH PERIOD 



Fig. 13. 



Comparative exponential rates of synthesis of glucose- 

 6-phosphate dehydrogenase, weight, volume and area by 

 OC and RS cells. 



100- - 



HSS IW 2W 3W IM 



Fig. 14. 



Differential solubilities of glucose-6-phosphate dehydro- 

 genase In 24 hr and 36 hr RS cells. 



have seen so far (Fig. 15). All assays were 

 done as in the foregoing experiment. The plot 

 in the figure shows that during the early stages 

 of exponential growth, most of the enzyme is 

 insoluble. But as the RS cell approaches the 

 end of its period of exponential growth, the % 

 soluble G6PDH gradually increases until, by the 



I00-- 



z 

 3 



< 

 o 



o 



o 

 a. 



10 



o 



80- 



60-- 



40-- 



20-- 



HRS. AT 24'>C. 

 12 24 36 48 60 72 



c^C 



«oO 



~i 1 1 1 1 r 



MORPH PT. 

 OF NO. RET 





Fig, 15. 



Changes In quantity and Isozymic composition of the 

 soluble G6PDH during ontogeny of an RS cell. 



time the point of no return in morphogenesis is 

 reached, essentially all of it is in soluble form. 

 This state of affairs persists for many hours 

 after the point of no return, but some "bound" 

 enzyme appears again as the RS cell approaches 

 maturity (i.e., 84 hours). So far, the data gen- 

 erally support the hypothesis shown in Fig. 12. 

 In order to obtain more definitive and infor- 

 mative data about the changes which occur in 

 this enzyme during cell differentiation, we have 

 begun to categorize its soluble and "bound" 

 forms via disc electrophoresis in polyacryl- 

 amide gel, using a TRIS-HCl-EDT A- Borate 

 buffer at pH 8.3. Although only a beginning has 

 been made, the patterns obtained (Fig. 15) for the 

 soluble enzyme reveal that striking changes in 

 its composition occur as the RS cell moves 

 along in its ontogeny. Two major bands (5 and 

 3) are present at 24 hours during exponential 

 growth, but as growth continues band 3 gradually 

 disappears and, by the time the point of no return 

 is reached, only band 5 remains. After the point 

 of no return, however, a new complex of bands 

 of G6PDH activity makes it appearance along 

 with band 5 which is present throughout develop- 

 ment. It is too early to speculate as to the role 

 of these isozymes in the bicarbonate trigger 

 mechanism, but we have every expectation, now, 

 that this approach will produce significant re- 

 sults. 



158 



