INTERMEDIATE REACTIONS IN PROTEIN SYNTHESIS I7I 



conipouiul containing the amino acid in this position. The stabihty of this compound 

 is not unHke that of amino acid-RNA. (Sec footnote 4 of text.) 



Dr. Beyer: Would you please discuss the recent findings of Drs. Moldave and 

 Meister, and of Fruton concerning the nonenzymatic incorporation of activated amino 

 acids into boiled proteins in connection with your findings of cofactors required for 

 enzymic incorporation. Which do you think is the physiological pathway? 



Dr. Stephenson: The nonenzymatic incorporation of amino acid adenylates is ob- 

 tained using relatively high concentrations of the adenylates. In the physiological sys- 

 tem, the activated amino acids are apparently enzyme bound and the free amino acid 

 adenylates are present in extremely low concentrations. Because of this and because of 

 the various nucleotide cofactors, such as CTP and GTP, rec|uired for the incorporation 

 of amino acid into RNA and protein, we assume that nonenzymatic reactions jilay a 

 very small role in our incorporation system. 



Dr. Marshak: Your data indicate attachment of the amino acid on the 2' or 3' carbon 

 of the ribose on the terminal adenylic or cytidylic acid group of the RNA. This site 

 differs from that of Michelson's in his hypothesis for RNA and amino acitl synthesis 

 as alternate pathways by polymerization after initial attachment of amino acid in 

 anhydride link to the 2 or 3' carbon of ribose of the nucleotide. Could you discuss 

 these findings? 



Dr. Stephenson: At the moment I am uncertain of the details of Michelson's paper 

 and cannot say how our data bear on this hypothesis. [The amino acid-RNA bond ap- 

 pears to be more stable than the 2':^ cyclic phosphate-amino acid anhydride bond 

 suggested by Michelson. However, we have not ruled out this possibility. The stability 

 of the AA-RNA is more like that of an amino acid ester which could conceivably be 

 on the 2' or 3' hydroxyl of the ribose. Since our data suggest that each amino acid 

 occupies a terminal position on an S-RNA chain, it would be difficult to see how 

 polymerization of the amino acids could take place using this RNA as a template. 

 Perhaps the microsomal RNA plays a role here.] 



Dr. Milkman: Are these techniques being applied to systems where ostensibly large 

 amounts of one protein are being made; for example, immature red blood cells' Do 

 you think the time is ripe for such applications of these techniques? 



Dr. Stephenson: Dr. Campbell has demonstrated the labeling of albumin in li\er 

 homogenates and the reticulocyte system is being studied by Drs. Rabinowitz and 

 Dintzis, and by Dr. Loftfield in our laboratory. Recently Dr. Loftfield has obtained 

 incorporation of C'"*-valine into ferritin isolated after incubation of a cell-free liver sys- 

 tem, using rats which had been stimulated to produce ferritin by in vivo injection of 

 iron oxide. This has been our first real evidence that our cell-free incorporation was 

 not just into large polypeptide or incomplete proteins which [irecipitated with tri- 

 chloroacetic acid. You are right, the time is ripe for such studies. 



