BIOCHEMICAL PROPERTIES OF THE ISOLATED NUCLEUS I95 



phorylation usually observed in mitochondrial suspensions. A few of the resem- 

 blances and some of the most striking; differences can be briefly summarized. 



fn the isolated nucleus, the synthesis of ATP requires oxygen and it is inhibited 

 by anaerobiosis, by 2, 4-dinitrophenol, sodium azide, sodium cyanide, antimycin A, 

 and Amytal. In all these respects it resembles ATP synthesis by mitochondria. On 

 the other hand, several inhibitors of mitochondral oxidative phosphorylation, in- 

 cluding carbon monoxide, calcium ions, Janus green P), methylene blue and 

 dicumarol, have little or no eflect on ATP synthesis by the nucleus (20). 



It is a matter of some interest to compare the effects of these different com- 

 pounds on ATP synthesis with their effects on nuclear amino acid incorporation. 

 Such a comparison is given in table 5. It is clear that agents which block ATP 

 synthesis in the nucleus also block amino acid uptake into its proteins. 



The evidence relating amino acid incorporation to ATP synthesis is not limited 

 to the inhibition studies summarized in the table. A more direct test of linkage 

 became possible when it was discovered that acetate ions at pH values below 5.9 

 remove nuclear ATP (20). A study of C'*-alanine uptake then showed that nuclei 

 exposed to acetate buffers over the pH range 4.4-5.9 were far less active than 

 'control' nuclei maintained at comparable pH values in other buffers which did 

 not extract nuclear ATP. The extent of C"-alanine incorporation was measured 

 by subsequent incubation of the 'control' and acetate-extracted nuclei in a neutral 

 medium (20). For example, it was found that nuclei which had lost 77 per cent 

 of their ATP had also lost over 90 per cent of their capacity to incorporate alanine- 

 i-C^*. 



The ATP requirement for protein synthesis by the nucleus is, of course, remi- 

 niscent of observations on cytoplasmic systems. The resourceful and stimulating 

 experiments of Hoagland, Keller, Zamecnik and Stephenson have shown that the 

 activation of L-amino acids prior to their incorporation into microsomal proteins 

 involves the formation of amino acyl-adenylate compounds. These compounds ap- 

 pear in reactions requiring specific enzymes and adenosine triphosphate (16, 15). 



Table 5. Efkects of metabolic; inhibitors on phosphorylation of nucleotides and 

 incorporation of amino acids in isolated thymus nuclei 



CHANGE IN CHANGE IN ALANINE- I -c'" 



PHOSPHORYLATION DUE UPTAKE DUE TO ADDED 



INHIBITOR CONCENTRATION TO ADDED INHIBITOR, ° o INHIBITOR, /i 



Sodium cyanide i X 10''' m — 100 — 76 



2, 4-dinitrophenol 2 X 10"' M — 100 ■ — 84 



Sodium azide i X 10 " ^^ — 100 — 9i 



Antimycin A 1 fig/m\ — 61 — 89 



Methylene blue 2 X i" " ^' +3° +3 



Methylene blue 2.5 X '" ' ^' +21 — 18 



Calcium ions 2 X 10" moo 



Calcium ions 3 X 10"" m o o 



Calcium ions 4 X i" ^' 



