KARASEK ET AL. 



Ill 



TRY 



TRY- AMP 



-6 



6 cm 



Fig. 2. Paper ionophoresis of tryptophan (TRY) and tryptophanyl adenylate (TRY- 

 AMP) in 0.05 M ammonium formate buffer (pH 4.5) ; apparatus of Markham and 

 Smith [11]. 



ganic pyrophosphate, and tris(hydroxymethyl) aminomethane buffer led to 

 synthesis of adenosine triphosphate. Adenosine triphosphate was identified by 

 coupling the reaction between radioactive inorganic pyrophosphate and trypto- 

 phanyl adenylate with the phosphorylation of glucose by hexokinase. The phos- 

 phate esters were separated by ethanol-barium salt fractionation, and radioac- 

 tive glucose-6-phosphate was identified by paper chromatography in two solvent 

 systems. 



An experiment designed to demonstrate the net synthesis of tryptophanyl 

 adenylate was carried out as follows. The total yield of tryptophan-activating 

 enzyme obtained from 10 lb of beef pancreas (15 mg) was incubated with 

 DL-tryptophan-3-C 14 , magnesium chloride, crystalline pyrophosphatase, adenosine 

 triphosphate, and tris (hydroxy methyl) aminomethane buffer for 30 minutes 

 at 37° C. At the end of the incubation period, 2 mg of synthetic tryptophanyl 

 adenylate was added as carrier and the reaction mixture was lyophilized. The 

 lyophilized reaction mixture was fractionated according to the scheme shown 

 in figure 3. 



In the first step of this procedure, treatment with glacial acetic acid in the 



J — " 



Enzyme ] 



ATP + TRY-C 14 +TRY-AMP 



v Ether 



ATP + TRY-AMP 



TRY-C 



14 



Paper electrophoresis 

 (/>H 4.5 and 0°) 



Fig. 3. Scheme for the isolation of tryptophanyl adenylate (TRY-AMP) from enzymatic 

 reaction mixtures. 



