April 5, 1954 



Cyclic Regeneration of Carbon Dioxide Acceptor 



1761 



and glucose phosphates by a series of reactions simi- 

 lar to a reversal of glycolysis. These conclusions 

 were supported by the observations that when car- 

 bon- 14 is administered to the photosynthesizing 

 plant as C'Oj, the first radioactive compound iso- 

 lated is carboxyl-labeled PGA, followed shortly by 

 dihydroxyacetone phosphate (DHAP), fructose 

 monophosphates (F.MP) and glucose monophos- 

 phate (GMP), both hexoses being 3,4-labeled. Af- 

 ter longer exposures of the plant to C'^Oj, radio- 

 carbon appears in other carbon atoms of PGA and 

 hexose and the distribution of activity is in agree- 

 ment with the above conclusions. 



•C 



I 



*c + c* 



•c 



I 



•c 



I 

 •c 



2[H| 



•c 



I 

 •c- 



I 

 •c 



PGA 



••c 



I 



••c 



I 



•c 



I 



•c 



hexose 



Observations on the rate and distribution of la- 

 beling of malic acid*"' showed it to be the eventual 

 product of a second carboxylation reaction which 

 is accelerated during photosynthesis, and it was 

 proposed that this second carboxylation played a 

 part in the reduction of carbon in photosynthesis, 

 leading eventually to the formation of the two-car- 

 bon CO2 acceptor (A, above). Malic acid, itself, 

 apparently was precluded as an actual intermediate 

 by inhibition studies,' but was thought to be an in- 

 dicator of an unstable intermediate which was 

 actually the first product of the second carboxyla- 

 tion. The discovery' of rapidly labeled sedoheptu- 

 lose monophosphate (SMP) and ribulose diphos- 

 phate (RDP) led to their inclusion in the proposed 

 carbon reduction cycle leading to the two-carbon 

 CO2 acceptor. 



The reciprocal changes in reservoir sizes of RDP 

 and PGA observed when algae were subjected to 

 light and dark periods' indicated a close relation- 

 ship, perhaps identity, between the RDP and the 

 two-carbon CO2 acceptor. 



In order to test these conclusions, it was neces- 

 sary to design experiments involving very short ex- 

 posures of the plant to C'*02. In some of these ex- 

 periments, the C* was administered during "steady 

 state" photosynthesis, the environmental condi- 

 tions (hght, carbon dioxide pressure, etc.) being 

 kept as nearly constant as possible for the hour pre- 

 ceding and the time during the experiment. Deg- 

 radation methods have been developed for sedohep- 

 tulose and ribulose and complete distribution of 

 radioactivity within these sugars obtained. 



The results of these experiments seem to obviate 

 the possibility that the second carboxylation reac- 



(4) A. A. Benson, S. Kawaguchi, P. M. Hayea and M. Calvin. This 

 Journal, 74, 4477 (1952). 



(5) A. A. Benson, et at., "Photosynthesis in Plants," Iowa State 

 College Press, Ames, Iowa, 1949, p 381. 



(6) D. W. Racusea and S. Aronoff, Arch. Biochem. Biophys., 42, 25 

 (1953). 



(7) J. A. Bassham. A. A. Benson and M. Calvin, J. Biol. Chem., 

 18», 781 (1950). 



(8) A. A. Benson, el at . ibid . 196, 703 (1952). 



(9) M. Calvin and Peter Massini, Expcrienlia, 8, 445 (1952). 



tion (leading to malic acid) is a step in carbon reduc- 

 tion during photosynthesis. Since no new evi- 

 dence has been found for the second "photosyn- 

 thetic" carboxylation, it would appear that a carbon 

 reduction cycle involving only one carboxylation 

 (leading to PGA) is more likely than the previously 

 proposed two-carboxylation cycle. 



Experimental Procedure 



Short "Steady State "Eiperiments. — Algae (Scenedesmus 

 obliquus) were grown under controlled conditions,' centri- 

 fuged from the growth medium, and resuspended in a 1% by 

 volume suspension in distilled water This suspension was 

 placed in a rectangular, water-jacketed illumination cham- 

 ber 6 mm. thick, through which was passed a continuous 

 stream of 4% COi-in-air (Fig. 1). From the bottom of the 

 chamber, a transparent tube led to a small transparent 

 pump constructed of appropriately placed glass valves and 

 two 5-cc. glass syringes mounted on a lever arm in such a 

 position that the syringe plungers moved in and out recipro- 

 cally about 5 mm. when the lever arm was moved back and 

 forth by a motor-driven eccentric. The output of the pump 

 was divided, the major portion being returned to the illu- 

 mination chamber and a smaller portion (20 ml. /minute) 

 forced to flow through a length of transparent "Transflex" 

 tubing of about 1 mm. diameter and thence into a beaker 

 containing boiling methanol. This solvent was found to 

 have an apparent killing time of less than 0.2 sec. as deter- 

 mined by the cessation of carbon fixation during photosyn- 

 thesis. The linear flow rate of algal suspension in the tube 

 was about 57 cm. /second. A solution of C'Oj in water 

 (0.0716 M, 110 MC./ml.) in a 30-cc. syringe was injected 

 through a fine hypodermic needle into the Transflex tubing 

 at a point a selected distance from the end of the tubing. 

 From the known flow rate of algal suspension in the Trans- 

 flex tubing and distance of flow from the point of injection 

 of C'*Oj to the killing solution, the time of exposure of the 

 algae to C'* was calculated. The flow of the C'Oj-contain- 

 ing solution was controlled by driving the syringe plunger 

 with a constant speed motor, and the flow rate was 0.5 ml./ 

 minute. The resultant dilution of the algal suspension was 

 2.5% and the increment in total CO2 concentration less than 

 15%. 



{hot pl>ti r 



Fig. 1. — Schematic diagram of flow system for short exposure 

 of algae to C"Oi. 



Since the flow of algal suspension in the tubing was not 

 turbulent, some difference in rates of flow at the center and 

 at the edge of the tubing was unavoidable. The extent of 

 this difference was approximately determined by injecting 

 a concentrated dye solution for about 0.5 sec. through the 

 hypodermic needle while the flow rate in the tubing was 20 



93 



