J. A. BASSHAM AND M. CALVIN 



splitting" would produce CO2 and a 3-keto pentose. Other 

 possibilities are hydrogenation of the carbonyl group followed by 

 a rearrangement which would result in one molecule of PGA 

 and one molecule of phosphoglyceraldehyde ; or a direct splitting 

 or "reverse benzoin" type reaction which would result in the 

 formation of one molecule of phosphoglyceraldehyde and one of 

 3-phosphohydroxypyruvic acid. 



Since PGA is known to be one if not the only product of the 

 carboxylation reaction in vivo, it is necessary to consider only 

 the "acid splitting" and reductive splitting. There was con- 

 siderable evidence for the acid split leading to PGA only, even 

 before the enzyme was studied in vitro. Studies were made of the 

 change in concentrations of RDP and PGA which occur in algae 

 immediately after turning off the light (18 and Bradley, private 

 communication). It was found that the concentration of PGA 

 rose very rapidly under these conditions while that of RuDP 

 dropped very rapidly. This was explained as resulting from a 

 cessation in the reduction of PGA due to the sudden decrease in 

 photochemically formed reducing agent, but at the same time, 

 a continuation of the carboxylation of RuDP leading to the 

 formation of PGA. The latter reaction, therefore, was not 

 apparently affected at once by the lack of illumination. This 

 result indicates that the formation of PGA from RuDP and CO2 

 does not necessarily involve a reduction, although it is possible 

 to postulate for this reaction a reducing agent with a longer half 

 life in the dark than the reducing agent required for the reduction 

 of PGA. 



Finally, recent work in this laboratory (J. Mayaudon, 

 private communication) with a somewhat more purified enzyme 

 preparation, and with C^^-labeled RuDP and CO2, indicates that 

 the only product of the carboxylation reaction in vitro is PGA. 



Despite these arguments, there still remains some possibility 

 that the first product of the carboxylation reaction, the six- 

 carbon j8-keto acid, might undergo different fates in the light 

 and in the dark. In the dark, splitting to two molecules of PGA 

 would proceed as discussed above; but in the light, with 



40 



