VIII. PROTEIN SYNTHESIS AND GENE ACTION 367 



of C^'^-tyrosine might be due to exchange of C^ --tyrosine with C^"*- 

 tyrosyl-RNA yielding dilution of the specific activity. Such an exchange 

 seems likely because of the long incubation (in the presence of ATP 

 and a generating system) which was used. On the other hand, the condi- 

 tions of the incubation, with a veiy high microsome: amino acyl-RNA 

 ratio, were such that transfer of a single amino acid might well have 

 been observed, in the relative absence of transfer of another. Bishop 

 and Schwect (1961c) fractionated the 105,000 (/ supernatant of the rabbit 

 reticulocyte lysate using salting out with ammonium sulfate, adsorption 

 to calcium phosphate gel, and chromatography on DEAE-cellulose; 

 they obtained two enzyme fractions which showed a 6- to 10-fold 

 enhancement of activity when assayed together. These fractions showed 

 small differences in relative activities in the transfer of four amino acids 

 under conditions (3-minute assay in the absence of ATP or an ATP- 

 generating system) which precluded the occurrence of a significant 

 exchange reaction between RNA-bound and free amino acids. Fessenden 

 and Moldave (1961), using rat liver ribosomes, have reported that the 

 enzymes removed from microsomes with deoxycholate did not catalyze 

 transfer unless a purified, heat-stable, but nondialyzable fraction of the 

 soluble cytoplasm was added. The significance of these fractions is not 

 apparent, since microsomes have been reported to contain transfer en- 

 zymes and to show full activity if glutathione alone is added (Hiilsman 

 and Lipmann, 1960). Thus, the fraction solubilized by DOC should 

 contain all the transfer enzymes. 



On the other hand, Takanami (1961), using a rat liver system, and 

 Nathans and Lipmann (1961) w^ith E. coli, have fractionated the super- 

 natant factor on DEAE-cellulose without observing any separation of 

 transfer activities. It appears likely that more than one enzyme is 

 involved in the transfer reaction. However, whether separate stages 

 in the reaction are catalyzed by different enzymes, or whether amino 

 acid-specific fractions have been separated is still not clear. 



3. The Mechanism of the Tran.sjer Reaction 



Evidence that transfer RNA is repeatedly recharged with amino 

 acids, after discharging bound amino acid on the template, was presented 

 by Allen and Schweet (1960). The complete reticulocyte cell-free system 

 (starting with free amino acid) showed a considerable requirement for 

 added transfer RNA which was fulfilled by 45-50 fig of RNA. The stimu- 

 lation of C^*-leucine incorjioration into protein, however, was 15-20 

 times as much as the amount of leucine required to charge completely 

 50 /xg of transfer RNA. Thus, the added transfer RNA must have re- 

 cycled many times in the course of the incu!)ation. Independently, 



