294 ALLEN, WHATLKY, llOSENBERG, CAPINDALE, ARNON 



you inaccuratelj^ quoted what I said, and I wish to correct that inaccuracy now be- 

 cause I don't want to be misciuotod asain. What I said hist year, and what I am 

 saying now, is that we are keeping an open mind (jn the path of carbon in photo- 

 synthesis. That does not mean that we are accepting or rejecting the present 

 concept. What we are saying is that, if the entire jjhotosynthetic process is localized 

 in chloroplasts, then we must find in tijcm finally a pathway for carbon fixa- 

 tion that would be the same as in the whole cell. That we have not done }'et. For 

 that reason we do not accept at this time the Calvin-Horecker pathway because 

 we don't as yet have the evidence that would permit us to say that this is the path- 

 waj^ of carbon fixation in the chloroplast. 



As for the point of adsorption of enzymes raised by Dr. Strehler, I think there is 

 a great deal of misunderstanding as to just what is being done in getting chloro- 

 plasts out of the cell. In the intact cells you have chloroplasts which are large 

 particles in relation to mitochondria. We separate chloroplasts because of their 

 large size by ditTerential ccntrifugation and we throw away everything else. So 

 any major contamination is removed initially. This is Point 1. 



Point 2. We wash these isolated chloroplasts once. 



Point 3. We add the remaining cytoplasmic constituents to the chloroplasts. 

 On the hypothesis that the active constituent in CO2 fixation and ATP formation 

 is some small contaminant remaining on the chloroplasts after differential centri- 

 fugation and washing, we would expect an increase in activity when we add all 

 the rest of the cytoplasm to the chloroplasts. However, no such effect is found. 



Point 4. The fixation of the COo is proportional, as you have seen on the slide 

 shown by Dr. Allen, to the chlorophyll concentration in the chloroplasts. 



Point 5. Directly in answer to Dr. Strehler's question — we C9n now take whole 

 chloroplasts, break them, thus removing this hypothetical coagulation barrier 

 which is supposed to trap extraneous enzymes, without losing any capacity for 

 ATP formation by the remaining chloroplast fragments. Capacity for CO2 fixation 

 is restored to these broken chloroplasts by the addition of a water extract of whole 

 chloroplasts. 



We feel that these five points render it extremely unlikely that there is any 

 significant enzymatic contribution from outside the chloroplast. 



Kok: We must distinguish our attacks on the problem. I do feel that it is not a 

 fair statement to say that Hill's publications have shown only that the chloro- 

 plast is something special. Hill has shown that the mechanism of photosynthesis, 

 can be broken apart, that the photolysis of water, which generates the reducing 

 power, can be fully separated from the process of carbon dioxide conversion. 



Amon : There is one simple point, Dr. Kok, that is very important. The cell is a 

 very large system. We have all kinds of enzymes in there. If you can be sure that 

 all the enzymes in photosynthesis are localized on one place in the cell, that at 

 once gives you an enormous simplification of the problem because you can look 

 for them in the chloroplast and you reduce the chance of confusing the respiratory 

 cycle or other metabolic cycles with photosynthesis. That is why these problems 

 of localization of cellular enzymes and their function are related. 



The first point in our approach was to localize the site of complete photosyn- 

 thesis in chloroplasts. What comes next is getting the enzj'mes out from the chloro- 



