1704 CHEMICAL PATH OF CARBON DIOXIDE REDUCTION CHAP. 36 



in darkness in photosynthetic bacteria) the energy of these bonds is used 

 up for the fixation and reduction of carbon dioxide. 



Wassink, Wintermans and Tjia (19510 made similar experiments with 

 Chlorella. In addition to measuring the inorganic phosphate in the me- 

 dium, the amount of phosphate in the cells extractable with trichloroacetic 

 acid (TCA) also was determined. In the absence of carbon dioxide, phos- 

 phate was taken up from the medium in light, and up to 30% of the TCA- 

 soluble phosphorus in cells was converted into TCA-insoluble form. The 

 shifts were much smaller in the presence of carbon dioxide. 



Wassink, Wintermans and Tjia (1951*) found that glucose had the 

 same effect on the phosphate transformation in Chlorella as carbon dioxide. 



Wintermans and Tjia (1951) described the liberation of the largest part 

 of the phosphate, made insoluble in TCA by illumination in the absence of 

 carbon dioxide, by hydrolysis in 1 N HCl. The (much smaller) amount of 

 TCA-insoluble phosphate, synthesized in the presence of carbon dioxide, is 

 divided about equally between a labile (i. e., HCl hydrolyzable) fraction 

 and a fraction that is stable in 1 A^ HCl at 100° C. 



Kandler (1950) also made experiments on the "phosphate level" of 

 Chlorella pijrenoidosa in darkness and in light. He determined the diffusi- 

 ble "inorganic" phosphate and the "TCA-soluble" organic phosphate. The 

 former declined by 20% in the first V2 min. of exposure to light, rose to a 

 peak at 1 min., and, after a second shallow minimum at about 3 min., set- 

 tled to a constant level, about 10% below that in darkness. Upon switch- 

 ing the light off, the level of TCA-soluble phosphate rose in about 2 min., 

 to a maximum about 30% above the steady value in light, then declined 

 again and assumed a constant level about 10% above that in light. These 

 "transients" show remarkable similarity to those observed in gas exchange 

 and chlorophyll fluorescence (chapter 33) and C* incorporation in certain 

 intermediates (this chapter, section 9). 



Kandler interpreted these variations in phosphorus content as evidence 

 of the participation in photosynthesis of high-energy phosphate (ATP) 

 and discussed two alternative mechanisms for its formation and utilization 

 in the photochemical process (c/. below). 



Simonis and Grube (1952) and Grube (1953) took up the study, first 

 attempted by Aronoff and Calvin (1948), of the incorporation of P(32) 

 tracer in different fractions of green cells in darkness and in light. Aronoff 

 and Calvin could find no uptake of P in the TCA-soluble fraction of 

 Chlorella in light (which would be a sign of increased concentration of or- 

 ganic phosphates, such as ATP). Kamen, Gest and Spiegelmann (cf. 

 Gest and Kamen 1948, Kamen and Spiegelmann 1948) observed, in light, 

 mainly a change in the TCA-insoluble P* fraction; the effect was de- 

 creased by HCN poisoning. Simonis and Grube resumed this study using 



