GLUCOSE -"C 



- * 



W' 



■777777777777777777Z^777^ 



„ G6PDH - GLUCOSE -"C 



GLUCOSE 



+ 



GLUC0SE-'''C 



MORPHOLOGICAL 

 REVERSAL 



-^ I 



''RELEASE 



INCREASE 



IN S.A. 



GLUCOSE-'Y 



Fig. 16. 



Hypothesis regarding effect of morphological reversal on 

 the specific activity of free glucose in an RS cell. 



I30-- 



IIO-- 



90-- 



70-- 



600-- 



400-- 



200- 



GLUCOSE - C, SPEC. ACTIV. 



I INCREASE IN S.A. OF FREE 

 1^ GLUCOSE DURING MORPH. 

 '' REVERSAL 



35 

 CPM(47r SCAN) DUE 

 TO GLUC0SE-'''C 



--I2 



) RELEASED INTO 

 ' POOL DURING 

 I MORPHOLOGICAL 

 ^ REVERSAL 



40 



45 



pM GLUCOSE/ ML. 

 EXTRACT 



■ / 



--6 . — . — • — / 



RELEASED INTO 

 POOL DURING 



X MORPHOLOGICAL 



) REVERSAL 



I I I I 



_L. 



I I I 



35 40 35 



HOURS 



40 



Some months ago, I also tried to approach 

 this problem in another way, arguing as follows. 

 If the notion outlined in Fig, 12 is valid, the 

 RS cell could be grown in such a fashion that 

 an unlabeled glucose pool could be created in it 

 and detectable some hours before the end of 

 exponential growth, i.e., before the point of no 

 return (cf. Fig. 16). At this point, glucose- i**C 

 could be fed to it, and the RS cell allowed to 

 continue its normal growth. If G6PDH were 

 functioning at or near the cell surface, differen- 

 tial localization of glucose- ^^ C and/or the im- 

 mediate products of its metabolism might also 

 occur near the cell surface. Presumably, some 

 of this labeled glucose (or glucose derivatives) 

 would also leak into, and mix with, the unlabeled 

 free pool. If, now, morphological reversal were 

 induced by removing the bicarbonate before the 

 point of no return (where, as seen previously, a 

 shift in isocitric and ketoglutaric dehydrogen- 

 ases does indeed occur), perhaps the surface 

 sites to which G6PDH was presumably loosely 

 bound would also be affected, thus releasing 

 soluble G6PDH. If this release of enzyme were 

 also to give rise to release of the glucose (or 

 derivatives) previously localized at these sites, 



Fig. 17. 



Effect of morphological reversal on the specific activity 

 of the free pool of labeled glucose in an RS cell. 



specific activity of the free pool of labeled glu- 

 cose in the cell would increase. 



The results of such an experiment are shown 

 in Fig. 17. Glucose- ^^C was introduced into a 

 synchronized culture of RS cells and the total 

 intracellular pool (labeled plus unlabeled) of 

 free glucose at successive stages in their 

 ontogeny was determined with the "Glucostat" 

 reagent. The results are shown by the continuous 

 line in the lower right hand figure. At 32 hr. 

 (i.e., before the point of no return) and at 40 hr, 

 (i.e., after the point of no return) samples of 

 these labeled cells were also transferred to 

 water for 3hr.; morphological reversal occurred 

 in the former but not in the latter. The changes 

 in the total intracellular pool of free glucose 

 associated with the successful and unsuccessful 

 morphological reversals are shown by the 

 arrows in the same figure. 



Samples of the glucose from each pool were 

 then purified extensively by way of paper and 



159 



