CASTELFRANCO, MEISTER, AND MOLD AVE 117 



pure on the basis of the hydroxamic acid-ferric chloride color reaction [11]. 

 The major impurities consist of adenylic acid and amino acid formed by hy- 

 drolysis of the anhydride during hydrogenation. This method has been suc- 

 cessfully applied to the following amino acids: glycine, alanine, valine, leucine, 

 isoleucine, (3-alanine, proline, phenylalanine, tyrosine, tryptophan, glutamine, 

 asparagine, threonine, methionine, and serine. Studies on the remaining natural 

 amino acids are in progress. 



Evidence for the proposed anhydride structure (fig. 1) includes the follow- 

 ing: (a) hydrolysis in alkaline solution yields equivalent quantities of adenylic 

 acid and amino acid; (b) reaction with hydroxylamine yields the correspond- 

 ing amino acid hydroxamates, which have been identified by paper chromatog- 

 raphy; and (c) paper ionophoretic study indicates that the aminoacyl adenyl- 

 ates have a net positive charge at pW 4.5. The possibility that the carboxyl 

 group of the amino acid may be linked to the adenylic acid moiety through 

 a group (e.g., 6-amino group of adenine) other than the phosphoric acid group 

 appears unlikely in view of the unusual reactivity of these compounds. 3 An 

 additional property of a-aminoacyl adenylates which has proved of value in 

 characterization is their reactivity in the presence of the tryptophan-activating 

 enzyme and inorganic pyrophosphate to yield adenosine triphosphate [3]. 



a-Aminoacyl adenylates are very labile in aqueous solution at values of pH 

 above 5.5. Thus, at pH 7.2 at 37° C, they exhibited half-lives of 5 to 10 minutes. 

 On the other hand, carbobenzoxyaminoacyl adenylates suffered only about 10 

 to 20 per cent hydrolysis in 2 hours at 37° at pH. 7.2. Acetyl adenylate and 

 benzoyl adenylate exhibit stability of approximately the same order as carbo- 

 benzoxyaminoacyl adenylates under these conditions. 



Preparation of glycyl-C 14 -adenylate and tryptophanyl-C 14 -adenylate made it 

 possible to study incorporation of the respective amino acid moieties into pro- 

 teins in systems previously employed for studies of amino acid incorporation. 

 The enzyme preparation was obtained as described by Zamecnik and Keller 

 [12]; it consisted of the supernatant solution (containing microsomes) ob- 

 tained by centrifuging a 25 per cent rat liver homogenate at 12,000^. This 

 preparation catalyzed the incorporation of amino acids into microsomal pro- 

 teins in the presence of adenosine triphosphate and an adenosine triphosphate- 



3 Additional evidence for the proposed structure has recently been obtained. Thus, we 

 have been able to convert carbobenzoxytryptophanyl adenylate with nitrous acid to the 

 corresponding inosinic acid derivative. The latter compound has also been prepared by 

 condensing inosinic acid with N-carbobenzoxytryptophan by the procedure described in the 

 text for anhydrides of adenylic acid. 



Acylation of the hydroxyl groups of ribose appears to be excluded. Thus, carbobenzoxy- 

 aminoacyl adenylates consumed theoretical quantities of periodate and, after reaction with 

 periodate, reacted with hydroxylamine to give the corresponding carbobenzoxyamino acid 

 hydroxamates. Paper ionophoretic study of the carbobenzoxyaminoacyl adenylates in borate 

 and other buffers was also consistent with the presence of free ribose hydroxyl groups; 

 the mobility of these compounds (and of adenylic acid) was greater in borate buffer than 

 in tris(hydroxymethyl) aminomethane buffer at pH 9.1. 



