639 



J. A. Bassham 



In the first of our more recent studies, we investigated the kinetics of 

 the labeling of ribulose diphosphate and of PGA in vivo . We found that from 

 the beginning of the period of steady state synthesis with 1^002 until satur- 

 ation of the intermediates, the specific raxiioactivity of PGA was always 

 considerably higher than that of RuDP (2). if the carboxylation of RuDP results 

 in the formation of two molecules of PGA, then the carboxyl group of one of 

 every two PGA molecules will contain the newly incorporated l^C. 



To test this model, we subtracted from the total PGA radiocarbon an amount 

 of I'^C radioactivity which would correspond to the radioactivity expected in 

 this carboxyl if the model were correct. Since this carboxyl group would rapid- 

 ly saturate if the model were correct, this involved subtracting 1/2 x 1/3 = 

 1/6 of the saturation level of radiocarbon in PGA after about 30 seconds. The 

 remaining 1^0 which would have to be derived from the RuDP carbon atctns was 

 compared with the I'^C in RuDP. The remaining, or residual, PGA carbon atcxns 

 were found to be labeled to a higher degree of saturation than the average of 

 the five atoms of RuDP, suggesting that this model (2 molecules of PGA per 

 carboxylation) was incorrect. 



Using a different model in which only one PGA molecule was formed, and in 

 which it was formed from the newly incorporated 1^^002 and carbon atoms 1 and 

 2 of RuDP, a similar calculation showed that the residual carbon atoms of PGA 

 were not labeled more rapidly than the average of the five RuDP carbon atans 

 until after 50 seconds. In this case there was no contradiction between model 

 and data even after 50 seconds, since we know that carbon atoms 1 and 2 of 

 RuDP are more quickly labeled than the carbon atans 3, 4 and 5 (D. 



Prom this data and reasoning, we concluded that the carboxylation of RuDP 

 leads to only one molecule of PGA in equilibrium with the PGA pool. The other 

 three carbon atoms fron RuDP appear to have been converted either to a form of 

 bound PGA not in equilibrium with the pool, or to sane other molecule. We 

 speculated that if the in vivo reaction were reductive, the other molecule 

 might be triose phosphate. 



It was noted earlier that the radioactivity in PGA does not alviays extra- 

 polate to 100^ at zero time (D. Scmetimes such extrapolation gives 10-15!S 

 I'^C in sugar phosphates at zero time. This finding suggests that it is not 

 only the three carbon moiety derived fron carbons 3,^^ and 5 of RuDP which may 

 not be in equilibrium with the PGA pool. It appears that seme of the PGA 

 labeled with the newly incorporated I'^C in the carboxyl group is also bound, 

 perhaps to an enzyme, and converted to sugar phiosphates without freely equil- 

 ibrating with the PGA pool. 



The experimental evidence suggesting that the newly incorporated l^C does 

 not all pass through the free pool of PGA praipted us to perform further 

 kinetic studies on the labeling of intermediates of the carbon cycle during 

 photosynthesis. Light-dark transient studies were performed (16) under condi- 

 tions of steady state photosynthesis. As in earlier light-dark transient studies 

 (14, 17) J the level of PGA rose and the level of ribulose diphosphate fell when 

 the light was turned off. However, under the more nearly steady state condi- 

 tions used in the more recent study, the concentration of fructose diphosphate 

 was higher than that of ribulose diphosphate and both diphospl-iates fell to 

 zero in concentration in the dark with equal rapidity. Sedoheptulose diphos- 

 phate concentration also dropped, and the sum of the drops of these three 

 diphosphates was not more than equal to the transient rise in PGA concentra- 

 tion. The levels of dlhydroxyacetone phosphate and of fructose-6-nhosnhate 



