CHEMICAL PATHWAYS 101 



incorporation was better and continued at a linear rate for about 15 min 

 (Siekevitz, 1952; Zamecnik and Keller, 1954); it was possible then to 

 obtain reproducible and reliable results. The factors required for incor- 

 poration and the interactions between several components of the system 

 could be studied more easily and with more confidence than before. 



It is in the microsomal fraction that the highest rate of incorporation was 

 observed, in homogenates of liver as well as in the liver of the living animal. 

 The microsomes alone were almost inactive, but they would incorporate 

 readily in the presence of both mitochondria and the soluble fraction 

 (Siekevitz, 1952). The function of the mitochondria in such a system is 

 actually limited to the regeneration of ATP, for they can be dispensed with, 

 provided substances able to furnish phosphate-bond-energy by anaerobic 

 processes are available (e.g. hexosediphosphate, phosphocreatin, phos- 

 phoenolpyruvate). Dialysis of the soluble fraction suppressed the incor- 

 poration, but this could be restored to a large extent by a supplement of 

 ATP and adequate metabolizable substrate to regenerate it (Zamecnik 

 and Keller, 1954). 



In later studies, Keller and Zamecnik (1956) observed that the fraction 

 of this supernatant which plays a part in amino acid incorporation is pre- 

 cipitated when the pH of the dialysed supernatant is adjusted to 5. This 

 fraction, often called 'pH 5 enzymes', is of course a very crude mixture of 

 many components which precipitate together at that pH. The incorpora- 

 tion system could then be reconstituted from the microsomal fraction, the 

 redissolved pH 5 precipitate, ATP and an ATP regenerating system. 

 Reprecipitation or treatment of the pH 5 fraction by the anion exchanger 

 Dowex-1 inhibited the system almost completely. Reactivation was 

 achieved by ATP plus a small amount of guanosinetriphosphate. This is 

 one more compound required for amino acid incorporation (Keller and 

 Zamecnik, 1956; Littlefield and Zamecnik, 1957). The pH 5 fraction then 

 received special attention. Hoagland (1955) had found that the supernatant 

 fraction of a liver homogenate catalyses the exchange of pyrophosphate 

 with ATP in the presence of several amino acids, and the formation of 

 hydroxamates of the amino acids in the presence of hydroxylamine, thus 

 indicating the presence of amino acid activation enzymes in this super- 

 natant. Association of these activities with the pH 5 precipitate obtained 

 from the crude supernatant (Hoagland et al., 1956), strongly suggested 

 that these activation enzymes are the constituents of the pH 5 precipitate 

 which are required for amino acid incorporation. 



The system at this stage seemed to consist of an energy source capable of 

 regenerating ATP, the activation enzymes which will cause the condensa- 

 tion of the amino acids with ATP, the microsomal particles which are the 

 place where newly-formed polypeptides are first observed, and guanosine- 

 triphosphate. In between the activation enzymes and the microsomal 



