638 



J. A. Bassham 



triose phosphate to be in rather rapid equilibrium with each other. However, 

 if the relation between phosphoglyceraldehyde and phosphodihydroxyacetone were 

 different from that which exists in glycolysis, it is possible that the label- 

 ing of the dihydroxyacetone phosphate pool would not be reflected into the 

 phosphoglyceraldehyde pool. Such a situation might exist if the phosphoglycol- 

 aldehyde moiety were bound to an enzyme and if its conversion to dihydroxy- 

 acetone phosphate resulted in a larger negative free energy change than is 

 associated with the conversion of free phosphoglyceraldehyde to dihydroxy- 

 acetone phosphate. 



The possibility that phosphoglyceraldehyde and perhaps erythrose-4-phos- 

 phate as well, may exist only in a form bound to the enzyme such as enzyrae-S- 

 CO-R is most attractive. It could explain why neither the triose phosphate 

 nor tetrose phosphate are normally seen as labeled intermediates during 

 studies of photosynthesis with 1^^002 . If the enzyme-bound phosphoglyceraldehyde 

 were unable to react with inorganic phosphate to make phosphoryl-phospho- 

 glycerate, as it does in glycolysis, the oxidation of triose phosphate to 

 phosphoglyceric acid could be blocked. Such a block might be most advantageous 

 to the photosynthetic mechanism, in that it would prevent reoxidation of new- 

 ly fonned sugar phosphates during short periods of darkness. I shall return to 

 this point later. 



It is also possible that the existence of such a bound form of phosphogly- 

 ceraldehyde, with a block towards its oxidation, could account for the report- 

 ed lack of aldolase in seme photosynthetic organisms (10,11,12). a reversal 

 of the condensation reaction leading to fructose-l,6-<ilphosphate would give 

 dihydroxyacetone phosphate and bound glyceraldehyde phosphate. Aldolase is 

 known to bind dihydroxyacetone phosphate (13) as a Schiff base. Conceivably 

 scxne organisms bind both triose phosphates so tightly that they can only be 

 liberated by the condensation reaction leading to fructose-l,6-diphosphate. 

 One might expect the pool of free dihydroxyacetone phosphate, commonly 

 observed in studies of photosynthesis in leaves and in Chlorella with -'■^C02, 

 to be missing fron such organisms. 



I would now like to focus attention on our mere recent kinetic studies. In 

 most of these experiments we establish a condition of steady state photosyn- 

 thesis in which all reactions are proceeding at a constant rate and in which 

 the intermediate pool sizes are maintained at constant levels ('^jO^'. We then 

 introduce I'^COp in a step function in such a way that the specific activity of 

 the added tracer comes immediately to its final value and is mintained there 

 during the course of the experiment. The addition of the tracer is acccmpanied 

 by no other environmental change. Saiiples may be taken immediately following 

 the addition of the 14C02 at frequent intervals and continuing through the 

 time when intermediates of the carbon reduction cycle have become "saturated" 

 with radiocarbon. 



From the initial slopes of the labeling curves upon introduction of 1^C02, 

 we can calculate rates of flow of I4c through specific intermediate pools. ^^ 

 From the level of radioactivity in specific compounds when they are "saturated 

 we can determine the concentration of the actively turning over pool, by 

 dividing the total radioactivity by the specific radioactivity of the 1^^002 

 which is maintained constant and is measured. Following "saturation" of inter- 

 mediates of the carbon cycle, we may vary sane environmental factor, such 

 as light or CO2 pressure, and follow the changes in concentration in the 

 actively turning over pools which are seen as changes in the total radio- 

 activities of these nools (l,2j3,lM jl5) . 



