April 5. 1954 



Cyclic Regeneration of Carbon Dioxide Acceptor 



1765 



low show to be labeled significantly at these short 

 times. This calculation is quite approximate, the 

 concentration of compounds involved being meas- 

 ured in experiments with algae photosynthesizing 

 under somewhat different conditions {i.e., 1% CO2 

 instead of 4%). However, such a calculation does 

 show more clearly the rapidity with which radio- 

 carbon is distributed among the principally labeled 

 carbon atoms and the difficulty in assigning an 

 order of precedence of labeled compounds on the 

 basis of labeling rates alone. 



The fact that compounds besides PGA have fi- 

 nite initial labeling slopes (which results in their 

 percentage activity not extrapolating to zero at 

 zero time) might be explained in several ways. One 

 possibihty is that during the killing time some of 

 the enzymatic reactions (in this case reduction of 

 PGA and rearrangement of the sugars) may not be 

 stopped as suddenly as others (the carboxylation to 

 give PGA) or may even be accelerated by the ris- 

 ing temperature prior to enzyme denaturation. 



Another explanation is that some of the labeled 

 molecules may be passed from enzyme to enzyme 

 without completely equilibrating with the active 

 reservoirs which are actually being measured. This 

 sort of enzymatic transfer of radiocarbon could 

 invalidate precedence assignments based on rates of 

 increase in specific activities since the reservoirs 

 would no longer be completely in the line of carbon 

 transfer. That the equilibration between reser- 

 voirs and enzyme-substrate complexes is rapid com- 

 pared to the carbon reduction cycle as a whole is 

 indicated by the fact that all the reservoirs become 

 appreciably labeled before there is an appreciable 

 label in the a- and /3-carbons of PGA, the 1-, 2-, 5- 

 and 6-carbons of the hexoses, etc. In any event, it 

 would appear to be safer to establish the reaction 

 sequences from qualitative differences in labeling 

 within molecules (degradation data) and changes in 

 reservoir sizes due to controlled changes in one en- 

 vironmental variable rather than from quantita- 

 tive interpretations of labeling rate data. 



Table I shows the results of degradations on sug- 

 ars obtained from the soybean series. The first 

 column shows the variation in labeling of carbon 



Table I 

 Radioactivitv Distribution in Sugars SEDOHEPTin.osE 



AND HeXOSE from SOYBEAN LEAVES 



Time, 

 sec. 



0.4 

 0.8 

 1..") 

 3.5 

 .■i.O 

 8.0 

 10.0 

 20.0 



300 



Sedum 



^ .Sedoheptiilose 



C-4 C-1.2.3 C-4.5,G C-7 C-2 



. Hexose 



C-1.7 C-6 C-1,2.3 C-4,.'j.6 



8 

 IS 

 24 

 20 

 29 

 24 

 28 

 21 

 14 

 12 



3."' 



4.'' 



3fi 



44 

 37 



.^7 

 fiO 



(i4 



47 

 48 



52 

 51 



35 12 



7 



12.5 

 12,5 



28 15 



-o 



TIMC (SECONDS), 



Fig. 5. — Distribution of radioactivity incorporated in 

 "steady state" photosynthesis with Scenedesmus: ©, sedo- 

 heptulose phosphate; 9, glucose phosphate; ®, dihydroxy- 

 acetone phosphate; O, fructose phosphate. 



since the carbon dioxide is depleted just prior to the 

 administration of C'Oa. Included in the table is 

 a complete degradation of a sedoheptulose sample 

 from Sedum speclabile grown in radioactive carbon 

 dioxide for two days (kindly supplied by N. E. 

 Tolbert, Oak Ridge National Laboratory). As- 

 suming this sample is uniformly labeled, its degra- 

 dation indicates the probable limits of accuracy of 

 the other degradations — about ± 10% of the ob- 

 tained value, mainly due to plating and counting 

 errors resulting from the low amount of radioactiv- 

 ity available for degradation. The five degrada- 

 tions on sedoheptulose make it possible to obtain 

 separate values for all the carbon atoms. Although 

 the carbon-fourteen labels of carbon atoms 1 and 

 were not determined in the case of the Scenedesmus 

 experiments, they were assumed small and approxi- 

 mated equal to carbon-fourteen labels found in 

 carbons 2 and 7, by analogy with the soybean leaf 

 experiments where the labels of all carbon atoms of 

 the sedoheptulose were determined. The label in 

 each carbon atom of the ribulose can be obtained 

 individually from the three degradations performed. 

 The distributions in Table II should be interpreted 

 as a clear qualitative picture of the position of the 

 radioactivity within the molecule rather than as a 



Table II 



Radioactivity Distribution in Compounds from Flow 

 Experiments (Algae) 



Glyceric 

 acid 



S2 



6 



6 



-5.4 Seconds- 



Fructose 



3 



3 



43 



42 

 3 

 3 



Sedohep- 

 tulose 



2 



2 

 28 

 24 

 27 



2 



2 



Ribu- 



11 



10 



09 



5 



3 



8 5 Seconds 



Sedohep- Ribu- 



tulose lose 



3 

 22 



11 



11 



04 



8 



5 



number four of sedoheptulose obtained from soy- 

 bean leaves exposed to C'''02 for very short periods. 

 These soybean leaf experiments are, of course, not 

 intended to represent "steady state" photosynthesis 



quantitative picture. Fewer points were taken in 

 this "steady state" flow experiment than in the 

 one described earlier in order to obtain more la- 

 beled sugar per point for degradation purposes. 



97 



