162 SUBCELLULAR PARTICLES 



tern such as phosphopyruvate and pyruvate kinase (43). The amino acids were 

 incorporated into the proteins of the microsome fraction, but some factors from 

 the 15,000 g supernatant fraction were also necessary. These factors could be 

 concentrated by bringing the 15,000 g supernatant fraction to pH 5.2 and re- 

 dissolving the resulting precipitate in medium buffered at pH 7.4 (43). This crude 

 enzyme fraction has been termed the 'pH 5 fraction.' With this system contain- 

 ing now the microsomes, pH 5 fraction and ATP, there was shown a require- 

 ment for small amounts of guanosine triphosphate (GTP) in addition to the 

 rather high ATP requirement already demonstrated (29). The over-all incorpora- 

 tion into microsome protein was inhibited by ribonuclease (i, 43). 



It should be pointed out that the incorporation of amino acids in the cell-free 

 system proceeds for 15-20 minutes only. At the end of this in vitro incubation 

 the distribution of labeled amino acids between the microsomal and supernatant 

 protein fractions is similar to that found after a 3-minute in vivo experiment. 

 Although net synthesis of protein has not been demonstrated, degradation studies 

 and characterization of the labeled protein provide evidence that the amino acids 

 are incorporated into a long polypeptide chain and, in one case, into the purified 

 protein, ferritin. The 'protein' which is isolated in the usual experiment is a 

 material which precipitates with trichloracetic acid (TCA) and has been 

 thoroughly extracted with hot TCA and various lipid solvents. The incorporated 

 amino acids cannot be removed from the protein fraction by dialysis; they do not 

 react with ninhydrin; and they appear in small identifiable polypeptides on partial 

 hydrolysis (30). 



These early investigations have indicated that the ribonucleoprotein particles 

 of the cell cytoplasm are the initial site of protein synthesis and that ATP, GTP 

 and some soluble factors are necessary. More recent studies have described sev- 

 eral enzymatic reactions which may be involved in the biosynthesis of protein. 

 Although none of these steps is implicated with absolute certainty, enough evi- 

 dence has been presented to enable one to speculate as to the possible role these 

 isolated reactions may play in the over-all reaction. 



The initial process appears to be the formation of an activated amino acid as 



indicated: 



AA + ATP + E^[AA~AMP]-E + PP 



The activating enzymes have received a great deal of attention (38, 19, 21, 12, 

 3, 11), and only a few of their properties will be discussed here. Briefly, an 

 amino acid adenylate is formed enzymatically in the presence of L-amino acids 

 and ATP with the elimination of pyrophosphate. This reaction is reversible and 

 the activated amino acid appears to be tightly bound to the enzyme as it has not 

 been isolated free in significant amounts from the incubation system. Activating 

 enzymes have been found in a vast number of tissues, and for all of the natural 

 amino acids. Some appear to be highly specific for an individual amino acid. 



