total radioactivity of each intermediate is measured, and, 

 when this is divided by the known specific radioactivity of 

 the entering CO2, the total number of carbon atoms of each 

 intermediate compound in the sample can be calculated. 

 Thus the number of moles per unit volume of algae of the 

 various intermediates of the actively photosynthesizing sys- 

 tem may be determined. This number of moles per unit 

 volume of plant material is an average concentration, since 

 the distribution of molecules in such a heterogeneous system 

 is not homogeneous. 



This determination of the concentrations of intermedi- 

 ates in vivo is an extremely valuable tool which has many 

 uses, but let us proceed for the moment with one particular 

 application. Having taken a sample of algae for later de- 

 termination of the concentrations of compounds, the experi- 

 menter turns off the light and proceeds to take a series of 

 samples of the algae as rapidly as possible, which is about 

 every 3 seconds. When the concentrations of compounds in 

 these samples are determined, any changes resulting from 

 turning off the light will be revealed. The two most striking 

 changes are found to be in the concentration of PGA, which 

 increases rapidly, and in the concentration of one particular 

 compound, ribulose diphosphate, which drops rapidly to 

 zero (16,20). 



The increase in PGA on turning off the light is expected. 

 The cofactors, derived from the light reaction, are necessary 

 for the reduction of PGA. The rapid drop in ribulose diphos- 

 phate, taken together with the fact that other sugar phos- 

 phates initially do not drop rapidly in concentration, indi- 

 cates that the formation of ribulose diphosphate from other 

 sugar phosphates requires a light-formed cofactor. This con- 

 clusion agrees with the fact that the known enzyme, which 

 converts ribulose-5-phosphate to ribulose- 1,5-diphosphate 

 (RuDP), requires ATP (Table 2). The drop in ribulose di- 

 phosphate, alone among the sugar phosphates, means that it 

 is being used up by a reaction that does not require light. 



Ribulose diphosphate, then, is used up by some reaction 



19 



