118 MICROSOMAL PARTICLES 



generating system. Thus, in the system of Zamecnik and Keller [12], we 

 observed 57 and 81 cpm/mg protein, respectively, with glycine-1-C 14 and trypto- 

 phan-3-C 14 . As is indicated in table 1, incubation of the enzyme preparation 

 with radioactive glycyl adenylate resulted in significant incorporation of isotope 

 into the protein subsequently isolated. When the anhydride was hydrolyzed 

 with alkali before study, significant incorporation was not observed. Further- 

 more, equimolar concentrations of radioactive glycine plus adenylic acid did 

 not lead to incorporation. It should be emphasized that the specific activities of 

 the C 14 -aminoacyl adenylates were 5 per cent of the values for the free amino 

 acids used by us in the system of Zamecnik and Keller. 



TABLE 1. Incorporation Studies 



Reaction Mixtures * cpm/mg 



Enzyme + Glycine-1-C 14 + Adenylate 0.1 7 



Enzyme + Glycyl-l-C 14 -adenylate 17.1 



Enzyme t + Glycyl-l-C 14 -adenylate 195. 



Enzyme t + Glycine-1-C 14 + Adenylate 1 .3 1 



* The reaction mixtures contained enzyme ( 1 ml) 

 and glycyl-C 14 -adenylate (2.5 /zmoles; 3.6 X 10 5 cpm) 

 in a final volume of 2.5 ml; incubated at 38° for 

 30 minutes. Similar results were obtained when 

 concentrations of aminoacyl adenylate from 10" 2 M 

 to 10~ 6 M were employed. 



f Enzyme heated at 100° for 10 minutes. 



Although these results appeared to be consistent with the hypothesis that 

 aminoacyl adenylates are intermediates in the incorporation of amino acids into 

 proteins, further experiments have raised the possibility that such incorpora- 

 tion may be explained in terms of nonenzymatic acylation of protein. Thus, 

 it was found that, when the enzyme preparation was heated for 10 minutes at 

 100° before incubation with C 14 -aminoacyl adenylate, the incorporation of 

 isotope into protein was considerably greater than with the unheated enzyme 

 preparation. In the experiments with heated enzyme, appreciable incorporation 

 of isotope did not occur with hydrolyzed anhydride preparations. Similar re- 

 sults have been obtained with tryptophanyl-C 14 -adenylate. 



With both heated and unheated enzyme preparations, the binding of the in- 

 corporated amino acid to protein was quite stable and could be released only 

 by the drastic acid hydrolysis required for the cleavage of peptide bonds. Thus, 

 with glycine-l-C 14 -labeled protein (heated and unheated), the quantity of free 

 amino acids and the percentage of isotope released as C 14 2 by ninhydrin in- 

 creased in parallel fashion during hydrolysis with 6 N HC1 at 105° over a 

 period of 16 hours. When the proteins labeled by incubation with glycyl-1-C 14 - 

 adenylate and with tryptophanyl-3-C 1 '-adenylate were treated with l-fluoro-2,4- 

 dinitrobenzene, followed by acid hydrolysis, dinitrophenylamino acid prepara- 

 tions were obtained which contained more than 70 per cent of the radioactivity 



