37. NUCLEIC ACID AND PROTEIN SYNTHESIS 391 



and the remaining polynucleotide exclusive of the terminal group. One, 

 thus far, obtains the tentative picture that at least part of and perhaps 

 all of the molecule enters the microsome with its attached amino acid. 

 This is consistent with the theoretical considerations dealt with at the 

 end of this chapter. 



Let us now consider the other components required in these transfer 

 reactions. The continued requirement for ATP in the absence of a need 

 to activate the amino acids is still unexplained. It could be argued that 

 since the system is crude and is not entirely free of activating enzymes 

 it is necessary to add ATP to prevent, by mass action, the reversal of 

 the reaction by which the RNA amino acid was originally formed. This 

 possibility seems unlikely since C 12 -amino acid does not interfere with 

 transfer. Hokin and Hokin 102 have obtained evidence that the transport 

 of protein across the microsomal lipoprotein membrane may be an ATP 

 dependent reaction. This might also be examined as an explanation of 

 the ATP requirement. Thus far, however, it remains a mystery. 



Even more puzzling is the system's requirement for GTP. This nucleo- 

 tide was found early by Keller and Zamecnik, 185 to be an obligatory com- 

 ponent of the over-all incorporation system. It was shown neither to be 

 involved in the activation of amino acids 113 nor in the attachment of amino 

 acids to sRNA. 117 It is, however, clearly a requirement in the rat liver 

 and ascites tumor sRNA amino acid to protein transfer systems, as w T e 

 have seen. The nucleotide does appear to be involved directly in the trans- 

 fer reaction itself and not in some later step, for it has been found to be 

 a requirement in the transfer to isolated ribosomes, free of membrane 

 component. 35 An interesting sidelight on the GTP requirement is the 

 finding of Chantrenne 186 that azaguanine is an effective inhibitor of protein 

 synthesis under conditions where there is no striking effect on over-all 

 RNA metabolism. Chantrenne suggests the possibility that such action 

 may be concerned with a step in the protein-synthesizing process involv- 

 ing GTP. At any rate, the GTP requirement of the system remains one 

 of its most beguiling mysteries! 



The soluble enzymic component — the S 4 fraction — of the sRNA amino 

 acid to the protein transfer system is still another mystery. As we have 

 said, this is the supernatant fluid obtained after a pH 5 precipitation of 

 a 100,000 g supernatant fraction of an ascites tumor cell lysate. 122 This 

 fraction contains amino acid activating enzymes (the bulk of which are 

 precipitated at pH 5), the terminal nucleotide addition system, and rela- 

 tively little RNA. Whether these known enzymic components are the 

 explanation for its requirement in the amino acid transfer to protein, or 



185 E. B. Keller and P. C. Zamecnik, J. Biol. Chem. 221, 45 (1956). 



186 H. Chantrenne, Rec. trav. chim. 77, 586 (1958). 



