166 Zamecnik et al. 



system (phosphopyruvate plus pyruvate kinase) may be 

 omitted; however, GTP (or GDP) is still required. 



An amino acid activating mechanism has recently been 

 found which is catalysed by the "pH 5 enzyme" fraction of 

 the rat liver protein in the presence of ATP (Hoagland, 1955; 

 Hoagland, Keller and Zamecnik, 1956). The reaction appears 

 to proceed as follows : 



ATP + amino acid + enzyme :^ (aminoacyl-AMP) enzyme 



4" pyrophosphate 



The evidence points to several separate activating enzymes, 

 rather than to a single enzyme or activation site capable of 

 activating all the amino acids. 



The role of GTP (or GDP) in this incorporation process is 

 puzzling. It has thus far been unable to substitute for ATP in 

 Hoagland's amino acid activation reaction. GTP has been 

 found to be an essential cofactor for the incorporation of all 

 six labelled amino acids tested (Keller and Zamecnik, 1956). 

 It is a very specific cofactor, the only active one among many 

 nucleoside tri-, di- and monophosphates tested. Through the 

 kindness of Dr. Waldo Cohn we were able to test the following 

 dinucleotides of guanine, obtained from partial hydrolysis of 

 RNA and possessing a free 3'-phosphate: GC, CG, GU, UG, 

 GA and AG. All of these compounds, in roughly • 1 [xm per 

 ml. concentration, were unable to substitute for GTP. 

 Addition of 0-25 mg. of AGUC ribonucleotide polymer, 

 kindly furnished by Dr. Severo Ochoa, to the in vitro test 

 system caused a slight inhibition of the incorporation. In 

 order to test for the GTP requirement, it is necessary to use a 

 pH 5 enzyme fraction washed quite free of endogenous GTP, 

 and to employ a microsome fraction prepared by centrifuga- 

 tion of a diluted 15,000 X g supernatant fraction of rat liver 

 homogenate (Keller and Zamecnik, 1956). 



Our present conception of the sequence of events in the 

 process of protein synthesis as observed in rat liver cytoplasm 

 is summarized in Fig. 2. The role of the ribonucleoprotein 

 particle is considered to be the sequentialization of activated 



