279 



H. E. Davenport 



TABLE 1 



The Effect of Successive Water Extractions of Pea Chloroplasts on 



Their Capacity to Photoreduce NADP and Metmyoglobin in the 



Presence of a Saturating Amount of Ferredoxin 



Rate of Reduction 

 Treatment of Chloroplast (/ixmoles/mg chlorophyll /h) 

 Material Metmyoglobin NADP 



As prepared in 0- 4 M sucrose 



0- 1 M tris-HCl, pH 7- 8 188 87 



Extracted with water: 



twice 220 39 



three times 265 25 



six times 302 1_1_ 



Reaction mixtures contained (in 3 ml. ) chloroplast material equivalent to 

 0- 03 mg chlorophyll; pea ferredoxin 0- 15 mg and (in /zmoles) NADP 0- 5 or 

 metmyoglobin 0- 025; tris-HCl buffer, pH 8- 0, 150; sodium chloride, 20. 

 Reaction measured as increase in extinction at 340 m/n (NADP) or 582 mfi 

 (metmyoglobin) after successive periods of illumination of 50,000 lux. Gas 

 phase air 20°. 



Once it was established that the active material could be extracted from 

 isolated chloroplasts it was found more convenient to prepare it from leaves 

 as a by-product in the routine preparation of ferredoxin either by the method 

 of Davenport and Hill^^) or of Tagawa and Arnon(5). In either case material 

 precipitated within the ammonium sulphate saturation limits 50-66% was re- 

 served for further purification. The active material was shown by the method 

 of DeLuca, Weber and Kaplan^S) to contain flavin adenine dinucleotide and it 

 will be referred to as "chloroplast flavoprotein. " 



Ferredoxin and chloroplast flavoprotein were examined as two variable 

 quantities required for the restoration of NADP reduction by protein-depleted 

 pea chloroplasts. Fig. 1 shows that, in a reaction mixture containing a rate- 

 saturating amount of ferredoxin the rate of photochemical reduction of NADP 

 was linearly related to the amount of added flavoprotein until the photochemical 

 system became saturated with respect to this second soluble factor at a reduc- 

 tion rate similar in magnitude to rates obtained with unwashed chloroplasts 

 where ferredoxin is the only soluble factor needed. The converse type of ex- 

 periment where ferredoxin was the variable factor is shown in Fig. 2. 



Independent evidence that a factor normally retained within the chloro- 

 plast is involved in NADP reduction was obtained by the selective inhibition of 

 this second factor by phenyl mercuric acetate (PMA) in situ . It has been 

 shown(3. 4) that mercurial compounds inactivate ferredoxin; their inclusion in 

 the assay mixture would not, therefore, be expected to inhibit metmyoglobin 

 and NADP reduction differentially. In order to localize the action of the heavy 



