172 Discussion 



We have not done definitive experiments in our laboratory, but I suspect, 

 from the few we have carried out, that RNA is being synthesized, since 

 i*C-labelled ATP makes its way into RNA during that same time. 



Spiegelman: Along these lines it seems to me very worth while, in 

 order to define the nature of this system, to enquire more closely into the 

 relation between nucleic acid synthesis and protein. As you know, in 

 the study of enzyme formation there seems to be no doubt that there is a 

 mandatory coupling between RNA synthesis and synthesis of the new 

 protein molecule. It would be interesting to see whether for example an 

 agent like hydroxyuridine would affect this incorporation phenomenon 

 as it does the synthesis of the protein molecule. Dr. Zamecnik, in your 

 in vivo incorporation experiments have you ever simultaneously 

 injected a complete mixture of amino acids, in addition to the labelled 

 one, to see whether there was any effect ? 



Zamecnik: We have injected a "quenching" dose of the inert amino 

 acid and then one has a drop off. 



Spiegelman: The point of that experiment is to see whether, if you 

 make protein synthesis much easier, your incorporation will also 

 increase. 



Zamecnik: There is an adequate intercellular supply of free amino 

 acids in the whole animal, isn't there? 



Spiegelman: There is a supply, but is it adequate? 



Zamecnik: But you can calculate that it is adequate for about 5-10 

 minutes of protein synthesis in the rat liver. 



Spiegelman: The yeast cell has a very high internal supply too but you 

 can stimulate protein synthesis in yeast cells immediately and consider- 

 ably by providing an external supply of amino acids. I should also like 

 to know whether you have tried a reconstitution experiment of your 

 ribonuclease-treated material. 



Zamecnik: Yes, we have tried this type of experiment ; it doesn't work. 



Pirie: Have you got any evidence of intermediate peptides of any 

 size, large or small? 



Zamecnik: No. My colleague Dr. Loftfield has been specially interested 

 in that question and has been studying ferritin synthesis in the rat liver. 

 If you give colloidal iron oxide to a weanling rat, the weanling rat 

 synthesizes 10 mg. of ferritin within the next two days, whereas pre- 

 viously he had none. That is a case of de novo synthesis of protein. 

 The specific activity of leucine or valine in the ferritin is approximately 

 the same as that of the intracellular free leucine or valine concentrations, 

 and Loftfield concludes that the ferritin molecule is not supplied with 

 any appreciable amount of peptide fragments from the large amount of 

 liver protein already existing, but that the ferritin molecule appears to 

 be formed from free amino acids exclusively. Furthermore, in the amino 

 acid activating system there is no evidence that amino acids can react 

 with an activated amino acid to form a peptide. 



I might mention that it is still possible to preserve a modicum of 

 doubt as to whether there is a real distinction between "exchange" and 

 "synthesis" in amino acid incorporation experiments. It is hard for us 

 to make a distinction in the animal system we use. We seem to be 



