CASTELFRANCO, MEISTER, AND MOLDAVE 119 



originally incorporated into the protein. Similar results were obtained with 

 heated and unheated enzyme preparations. 



We have also found that, when glycyl-l-C 14 -adenylate is incubated with puri- 

 fied rat-liver ribonucleic acid obtained by phenol extraction [13], considerable 

 radioactivity remains associated with the ribonucleic acid preparation after ex- 

 haustive dialysis and ethanol precipitation. Approximately 40 per cent of the 

 radioactivity of the ribonucleic acid preparation was alkali-labile. When such 

 ribonucleic acid preparations were incubated with unheated and heated pro- 

 tein preparations, significant quantities of radioactivity were found in the protein 

 subsequently isolated. Thus, incubation of 1 micromole of glycyl-l-C 14 -adenyl- 

 ate with 50 mg of liver ribonucleic acid in 1 ml of water for 30 minutes at 

 38° gave a ribonucleic acid preparation containing 3000 cpm (after dialysis 

 and precipitation). When 20 mg of this C 14 -ribonucleic acid (1500 cpm) was 

 incubated with enzyme preparation (table 1) for 30 minutes at 38°, approxi- 

 mately 100 cpm was associated with the protein subsequently isolated. Similar 

 results were obtained with heated enzyme preparation. 



The observed reactions of the aminoacyl adenylates with proteins are consist- 

 ent with the reactivity expected of anhydrides of this type. A similar result might 

 occur when proteins are treated with radioactive acetic anhydride. The reaction 

 of the aminoacyl adenylates with the heated protein preparations would appear 

 to be a nonenzymatic acylation reaction involving the free reactive groups of 

 proteins. Heat denaturation of the protein would be expected to expose a greater 

 number of amino groups to the action of the acylating agents. 4 The extent to 

 which the labeling observed with unheated proteins may be due to an enzy- 

 matic mechanism is not known. Although we believe that in the present studies 

 transfer of the amino acid moieties from aminoacyl adenylates to proteins oc- 

 curred largely by a nonenzymatic process, the possibility cannot be excluded 

 that some of this transfer is enzymatically catalyzed. Perhaps nonenzymatic 

 acylation of proteins may also take place to some extent when amino acids are 

 incubated with adenosine triphosphate, an adenosine triphosphate-generating 

 system, and a suitable enzyme preparation. The aminoacyl adenylates formed 

 in the activation reaction [3] might be expected to react in such a manner. 

 Thus, the high reactivity of aminoacyl adenylates with protein and ribonucleic 

 acid may explain at least some of the reported [4, 12] incorporation phenomena. 

 It must be emphasized that the concentrations of aminoacyl adenylates pre- 

 sumably formed in the amino acid incorporation systems previously studied 

 would be expected to be considerably lower than the concentrations of aminoacyl 

 adenylates we have used. Much lower concentrations of aminoacyl adenylates 

 than those used here must be employed to make a meaningful comparison 

 of the labeling in the two systems. Such studies will require aminoacyl adenyl- 

 ates of considerably higher specific radioactivity. Although the present in- 

 vestigations raise the possibility that incorporation in cell-free systems into 



4 Porter [14] has reported that heat denaturation of several proteins increases the number 

 of e-amino groups of lysine that can react with acylating agents. 



