NUCLEAR PROTEIN SYNTHESIS 263 



ing Other large polyanionic molecules for it) restores nuclear ATP syn- 

 thesis; as a result, other energy- dependent synthetic reactions in the 

 nucleus can resume [14]. 



These two facets of amino acid incorporation in the intact isolated 

 nucleus (sodium ion requirement and DNA-dependence) set it apart from 

 protein synthesis in the cytoplasm, which requires potassium, not sodium 

 ions [15], and is sensitive to ribonuclease [16] and not to deoxyribonuclease. 

 Yet a detailed study of the mechanism of protein synthesis in sub-nuclear 

 fractions leads to the conclusion that the protein synthetic pathway in the 

 nucleus is essentially the same as that found in the cytoplasm (and previously 

 discussed in this symposium by Drs. Zamecnik, Hultin, Siekevitz, and 

 Campbell). 



AMINO ACID ACTIVATION 



In the nucleus, as in cytoplasmic and bacterial systems, the primary 

 step in protein synthesis appears to be an activation reaction requiring the 

 participation of ATP. The reaction is mediated by activating enzymes 

 similar to those originally described by Hoagland [17], according to the 

 equation : 



amino acid + ATP + activating enzyme?=^enzyme-AMP~ amino acid + PP 



A convenient method of assay for this activity is based on the reverse re- 

 action, in which pyrophosphate is incorporated into ATP. Using ^^p. 

 labelled pyrophosphate and measuring the production of radioactive ATP 

 [18] it was possible to show that extracts of isolated thymus nuclei will 

 form ^-ATP, but only if amino acids are present in the incubation mixture 



[19]; 



The ATP-pyrophosphate exchange reaction is promoted by the 

 addition of mixtures of amino acids or by individual amino acids tested 

 separately. Neutral extracts of highly purified thymus nuclei contain 

 activating enzymes for at least fifteen L-amino acids; the D-isomers of 

 these amino acids are not acted upon (Table I). 



Many of the activating enzymes can be precipitated from solution by 

 lowering the pH of the nuclear extract to 5-2, as was observed previously 

 in cytoplasmic systems. 



Many tests have been made to verify that the amino acid activating 

 activity is actually present in the cell nucleus and is not an artefact due to 

 the adsorption of cytoplasmic enzymes during the isolation procedure. The 

 most reliable evidence in this connection is that obtained from a study of 

 nuclei isolated in non-aqueous media by techniques which preclude any 

 exchange of water-soluble enzymes between nucleus and cytoplasm [20, 21]. 

 Two types of "non-aqueous" nuclei were selected for analysis because 

 their purity had been established by chemical, enzymic, and immunological 

 tests for cytoplasmic contamination ; these were the nuclei prepared from 



