RELATIONSHIP BETWEEN REDUCING FACTOR AND REDUCTASE 



451 



protein used here had been further purified by electrophoresis on paper. 

 With either hydrogen acceptor saturation of the chloroplast system 

 occurred at about the same concentration of added protein and at this 

 saturation level TPN was i • 3 times as effective as metmyoglobin in terms 

 of hydrogen equivalents transferred. This ratio, in different experiments, 

 was found to varv from 1-2 to 1-7 but the variation could not be related 

 to the method used in preparing the leaf protein. 



300 



200 



1 2 



mg protein added 



03 



Fig. 2. Comparison of the activity of pea leaf protein (PPNR further purified 

 by electrophoresis) in catalyzing the reduction of TPN and metmyoglobin. 

 Reaction mixture contained (in 3 ml.) leaf protein as indicated, spinach chloro- 

 plasts (chlorophyll 0-115 irig-), and (in //moles) phosphate buffer pH 77, 90; 

 NaCl, 40; and the following: TPN, 04; ADP, 05; MgCl.,, 15; • metmyo- 

 globin, 0-26. Leaf protein was omitted from the blank cells. 



Stimulation of TPN reduction by photophosphorylation 



The reactions shown in Figs i and 2 where TPN was the hydrogen 

 acceptor, were carried out in the presence of adenosine diphosphate (ADP), 

 orthophosphate and magnesium chloride. The presence of this phosphate 

 acceptor system was found to be essential for maximum reduction rates 

 provided that the chloroplasts were at, or near, saturation with respect to 

 added leaf protein. At saturation the additional phosphate-accepting 

 ingredients stimulated the reduction rate 2-5-fold. It was confirmed that 

 inorganic phosphate was incorporated as ATP in the molecular ratio 

 I TPNHo I ATP [7]. This resuh is at variance with the report of Jagen- 

 dorf [8] that no such stimulation of the rate of reduction of TPN during 

 phosphorylation had been detected in three laboratories in the United 



