112 MICROSOMAL PARTICLES 



cold separated tryptophanyl adenylate from the enzyme which remained in 

 the insoluble residue along with some magnesium chloride and tris(hydroxy- 

 methyl) aminomethane buffer. Subsequent addition of ether to the acetic acid 

 extract resulted in the precipitation of adenosine triphosphate and tryptophanyl 

 adenylate, leaving radioactive tryptophan in the supernatant solution. The pre- 

 cipitate, which contained both adenosine triphosphate and tryptophanyl adenyl- 

 ate, was washed several times with cold ether-glacial acetic acid followed by 

 several ether extractions in order to remove residual acetic acid. The precipi- 

 tate was dissolved in a small amount of ammonium formate buffer (pW 4.5), 

 and an aliquot of this material was analyzed ionophoretically. The paper 

 ionophoretic separation yielded a major radioactive band which corresponded in 

 mobility to authentic tryptophanyl adenylate. A smaller band, negatively 

 charged, was detected but has not yet been identified. The positively charged 

 band was eluted, treated with hydroxylamine, and chromatographed on paper 

 in several solvent systems. Radioactive tryptophan hydroxamic acid was identi- 

 fied in each system. 



A similar experiment carried out without the addition of carrier trypto- 

 phanyl adenylate was also performed. Ionophoretic analysis again revealed evi- 

 dence for the formation of tryptophanyl adenylate; however, somewhat less 

 radioactivity was found in the tryptophanyl adenylate area. These findings sug- 

 gested that there was incorporation of radioactive tryptophan into tryptophanyl 

 adenylate in the experiment with carrier. In order to investigate this possi- 

 bility directly, radioactive tryptophan was incubated with tryptophanyl adenyl- 

 ate, enzyme, and magnesium ions. The tryptophan hydroxamate isolated 

 from this reaction mixture (after the addition of hydroxylamine) contained 

 appreciable radioactivity; the findings therefore suggest that an exchange be- 

 tween tryptophan and tryptophanyl adenylate occurred : 



TRY-C 1 4 + TRY-AMP -» TRY-C 14 -AMP 4- TRY 



We have also found that the tryptophan-activating enzyme catalyzes the 

 formation of adenosine triphosphate from inorganic pyrophosphate and a va- 

 riety of a-aminoacyl adenylates. Thus, aminoacyl adenylates of l- and D-trypto- 

 phan, l- and D-phenylalanine, L-isoleucine, L-glutamine, L-alanine, glycine, L-pro- 

 line, L-valine, L-leucine, and L-tyrosine gave adenosine triphosphate in this 

 system. It is of some interest that d- and L-tryptophanyl adenylate and d- and 

 L-phenylalanyl adenylate were about equally active. Examination of the D-anhy- 

 drides (after hydrolysis) by optically specific enzymatic methods [12] revealed 

 that the optical purity of the amino acid moieties was greater than 99.5 per cent; 

 it is therefore unlikely that the activity of the D-aminoacyl adenylate is due to 

 the presence of adenylate derivatives of the corresponding enantiomorphs. Of 

 the aminoacyl adenylates examined, only those of a-amino acids were active. 4 



4 We have recently found that L-tryptophanyl inosinate [10] is inactive, and that inosine 

 triphosphate is not active in place of adenosine triphosphate in the forward reaction (reac- 

 tion 1, table 2). 



