DANIEL I. ARNON 551 



TABl.K ir. 



CO2 Fixation by a Chlorophyll-free Extract and a Complete 

 Chloroplast System from Spinach 



(Trebst, Tsujimoto, and Arnon, 153) 



C'Oi fixed 

 Treatment counts/minute 



1. Chlorophvll-free extract, dark, + A. P.' 134,000 



2. Chlorophyll-free extract, dark, -A. P.' 9,000 



3. Complete chloroplast system, light 200,000 



4. Complete chloroplast system, dark 20,000 



' A.P. = Assimilatory power, i.e., TPNH.> and ATP, formed in the light by 

 chloroplasts. 



The complete chloroplast system contained in a final volume of 2.5 ml, "broken" 

 chloroplasts (Cis) containing 0.5 mg chlorophyll, ciilorophyll-free extract (CE) equiv- 

 alent to 2 mg of chlorophyll and the following in micromoles: Tris buffer, pti 7.5, 80; 

 MgCli, 5; MnCfj, 2; Na ascorbate, 10; glucose-1 -phosphate, potassium salt, 0.3; 

 sodium phosphate, pH 7.5, 5; ADP, 2; and TPN, 2. The reaction was carried out at 

 20"C for 30 minutes; gas phase, argon. In Treatments 3 and 4, 10 micromoles NaHCOa, 

 containing C'^O-:, were injected at the beginning and the reaction vessels were illumi- 

 nated or left in the dark, respectively. In Treatment 1, the reaction mixture was first 

 illuminated without the addition of bicarbonate. After 30 minutes, the chlorophyll- 

 containing particles were removed by centrifugation, and the chlorophyll-free super- 

 natant fluid was incubated in the dark for 30 minutes with 1.5 micromoles cysteine and 

 10 micromoles of NaHCOs, containing C'^02. The same procedure was followed in 

 Treatment 2 as in Treatment 1, except that ADP and TPN were omitted. 



The products oi COo assimilation were found to be the same 

 whether CO2 assimilation occurred during continuous illumination 

 (Fig. 24) or in the dark, at the expense of assimilatory power gen- 

 erated during a preceding light period (Fig. 25) . The products in- 

 cluded hexose and pentose mono- and diphosphates, phosphoglyceric 

 acid, dihydroxyacetone phosphate, and small amounts of phosphoenol- 

 pyruvate and malate. 



Similar products of CO2 assimilation by chloroplast extracts, in- 

 cluding the formation of phosphorylated sugars, were obtained in a 

 total dark chemosynthesis where TPNH2 and ATP were not formed 

 photochemically but were prepared either chemically or enzymatically, 

 or were derived from animal material (153). 



An important link in the chain of evidence connecting the photo- 

 synthetic events with the dark assimilation of COo in chloroplasts 

 was provided by experiments showing that both cyclic and non-cyclic 

 photophosphorylation were essential. The ATP formed in non-cyclic 

 photophosphorylation alone (Equation 5) is instifficient for the as- 

 similation of COo to the level of carbohydrates (151) . 



Experimental evidence for the participation of cyclic photophos- 

 phorylation in COo assimilation by chloroplasts had to be sought in 



