[15. XII. 1952] 



M. Calvin and P. Massini: The Path of Carbon in Photosynthesis 



457 



HO,P-OCH,-CHOH-CHOH-CO-CH,-0-PO,H 



CO,. 2[H] 



►■ 



CO, 



HO,P-OCH,-CHOH— CHO 

 phosphoglyceraldehyde 



-HOjP— OCHj— CHOH— COOH 

 phosphoglyceric acid 



-> 2x-HOjP— OCHj-CHOH-COOH 

 ^^^^ phosphoglyceric acid 



first minute of darkness is actually higher than it is in 

 the steady state photosynthesis. This would be the case 

 if the C3-C2 cleavage of ribulose diphosphate, which in 

 photosynthesis presumably yields a triose phosphate 

 molecule beside the Q carbon dioxide acceptor, in the 

 dark yelds a molecule of phosphoglyceric acid instead 

 of the triose molecule. The overall reactions may be 

 represented above (not a mechanism). 



This hypothesis is supported by the fact that the 

 triose phosphate also decreases in the dark. 



The fact that the net result of the reaction sequence 

 in the light from ribulose diphosphate to phospho- 

 glyceric acid and triose phosphate is a reductive car- 

 boxylation and thus the reversal of the oxidative 

 decarboxylation which, in the case of pyruvic acid, 

 requires the presence of a cyclic disulfide compound 

 leads to the idea that the former sequence might be 

 catalyzed by a similar enzyme. This idea seems to be 

 supported by the results of an experiment performed 

 in this laboratory some time ago, which were difficult 

 to explain'. 



In order to examine the relation between photosyn- 

 thesis and the glycolytic cycle, a series of experiments 

 similar to those described previously were performed 

 with added iodoacetamide which is known to inhibit 

 the action of triose phosphate dehydrogenase', pre- 

 sumably through a reaction with its sulfhydryl group'. 

 A 1% suspension of Chlorella in phosphate buffer was 

 allowed to photosynthesize in light of 2500 footcandles 

 and an atmosphere of 1% carbon dioxide, 5% oxygen 

 and 94% nitrogen. At various times before adding the 

 radioactive bicarbonate solution, iodoacetamide was 

 added to give a 1-5 x 10"* M solution. 1 min after 

 adding the radiocarbon, the cells were killed and 

 extracted. 



After 8 min contact with iodoacetamide, the cells 

 were still able to fix 75% as much carbon dioxide as 

 non-poisoned cells otherwise treated the same way 

 (control). The amount of radioactivity in phospho- 

 glyceric acid was 50% of the control, and the amount 

 in sucrose had reached a sharp maximum of 3-5 times 



' W. Stepka, Thesis University of California (June 1951). 



» O. Meyerhof and W. Kiessling, Biochem. Z. 28/, 249 (1053). 



' I.. Rapkins. C. r. See. Biol. (Paris) HJ, 1294 (1933), 



that in the control. There was practically no radio- 

 activity in the ribulose diphosphate. After 90 min of 

 exposure to the poison the cells had practically lost 

 their ability of photosynthesis. 



If, in the proposed photosynthetic cycle, the cleav- 

 age of the heptose and pentose phosphates is depen- 

 dent on an enzyme containing sulfhydryl groups, which 

 were more sensitive to iodoacetamide than the triose 

 phosphate dehydrogenase, a picture similar to the one 

 described would be expected : After short exposure to 

 the poison, in relatively low concentration, the lack of 

 Cj carbon dioxide acceptor would slow down the photo- 

 synthetic cycle. The synthesis of carbohydrates, how- 

 ever, would proceed almost without inhibition, thus 

 decreasing the concentrations of the intermediates in 

 the cycle. This would allow the compounds to reach a 

 higher specific activity during the period of exposure 

 to radiocarbon (cf. equation (2), change of specific 

 activity inversly proportional to concentration]. At 

 some time after administration of the poison, the su- 

 crose would be labeled faster than in the control due to 

 the higher specific activity of its precursors. After a 

 longer period, however, the rate of synthesis of sucrose 

 would decrease because the pool of its precursors would 

 be exhausted. 



Zusammenjassung 



Die Trennung des Phanomens der Photosynthese 

 griiner Pflanzen in eine Lichtreaktion und die vom Licht 

 unabhangige Reduktion der Kohlensaure warden di.s- 

 kutiert. 



Die Reduktion der Kohlensaure und das Schicksal des 

 assimilierten Kohlenstoffs wurden untersucht mit Hilfe 

 der Spurenmethode (Markierung der assimilierten Koh- 

 lensaure mit C") und der Papierchromatographie. Ein 

 Reaktionszyklus wird vorgeschlagen, in dem Phosphogly- 

 zerinsaure das erste isolierbare Assimilationsprodukt ist. 



Analysierung des Extraktes von Algen, die in einem 

 stationaren Zustand fiir langere Zeit radioaktive Kohlen- 

 saure assimilierten. lieferte weitere Auskunft iiber den 

 vorgeschlagenen Zyklus und gestattete, die am Zyklus 

 beteiligten Mengen einiger Substanzen ungefahr zu be- 

 stimmen. Die friihere Vermutung. dass Licht den Res- 

 pirationszyklus beeinflusst, wird bestatigt. Die Moglich- 

 keit der Mitwirkung von a-Liponsaure (a-lipoic acid) oder 

 einer verwandten Substanz, bei diesem Effekt und im 

 Photosynthesezyklus, wird erortert. 



91 



