NUCLEAR PROTEIN SYNTHESIS 27 1 



sodium is particularly large in the case of alanine uptake [46] but it is easily 

 demonstrated for other amino acids as well. Lithium is partly effective as 

 a sodium substitute, but caesium, rubidium or potassium are without 

 effect. 



A number of quaternary ammonium ions were also tested because 

 Lorente de No [47] had shown them to be effective sodium substitutes in 

 maintaining membrane potentials in isolated nerve. However, neither 

 hydrazinium, guanidinium, or tetraethyl ammonium ions could promote 

 alanine incorporation into the proteins of sodium-deficient thymus nuclei. 



The reason for the sodium-dependence of the incorporation process 

 was sought for but could not be found in studies of the individual reactions 

 in the nuclear synthetic pathway. There was no specific sodium ion require- 

 ment for amino acid activation, or for transfer to nuclear RNA, and a 50*^0 

 reduction in sodium ion concentration had no effect on amino acid in- 

 corporation into isolated nuclear ribosomes [44]. This led us to test the 

 possibility that sodium ions might influence the rate at which amino acids 

 enter the free amino acid "pool" within the nucleus. 



This approach has not only provided an explanation of the sodium ion 

 effect but it has also revealed the existence of a mechanism, hitherto un- 

 known, for amino acid transport and accumulation within the cell nucleus. 



The penetration of free amino acids into the nuclear "pool" has been 

 measured as follows : nuclei w'ere suspended in a buffered sucrose medium 

 containing sodium (or potassium ions) and a small, accurately measured 

 amount of the radioactive amino acid. The mixture was incubated aero- 

 bically, with shaking. Aliquots of the suspension were removed at zero 

 time and at appropriate time intervals thereafter. The nuclei were centri- 

 fuged down, washed with sucrose, and then extracted wdth cold 2% 

 HCIO4. The radioactivity in each extract was measured and, after correc- 

 ting for the small amount of ajiiino acid adsorbed at zero time, the counts 

 were plotted as a function of the time of incubation. The time course of 

 [i-^'*C]-alanine transport at 37° is shown in Fig. 5, which compares the 

 radioactivity present in the nuclear "pool" after incubation in 0-04 M 

 NaCl with that observed at an equivalent concentration of KCl. The 

 transport of the amino acid into the acid-soluble "pool" is evidently 

 sodium-dependent, and does not occur to any appreciable extent when 

 equivalent amounts of potassium replace the sodium. There is good 

 evidence that the radioactivity in the perchloric acid extract of the isolated 

 nuclei occurs as free P^C] -alanine. This was checked by chromatography 

 of the concentrated extract on paper and subsequent elution of the 

 alanine spot. A definite increase in the radioactivity of the isolated alanine 

 is evident after only a few minutes' incubation. Other tests have shown 

 that, after 30 min. at io°C., the nuclei build up an alanine concentration 

 that may exceed the alanine concentration of the surrounding medium [46]. 



