196 SUBCELLULAR PARTICLES 



A search for similar activating enzymes in isolated nuclei is now under way, and 

 preliminary experiments indicate that activated amino acids can be isolated from 

 nuclei as the hydroxamates (17). 



ROLE OF POLYNUCLEOTIDES IN NUCLEAR ATP SYNTHESIS 



The experiments now to be described relate the DNA of the nucleus directly to 

 the synthesis of adenosinetriphosphate. The first of these is the demonstration 

 that nuclei pretreated with deoxyribonuclease lose their ability to synthesize ATP 



(fig- 5)- 

 In this experiment nuclear suspensions were incubated for 30 minutes at 38° 



in the buffered sucrose-NaCl medium used in amino acid incorporation experi- 

 ments (2). Crystalline pancreatic deoxyribonuclease was added to half of the 

 flasks; nuclei in the other flasks served as 'controls.' After incubation the nuclei 

 were centrifuged down and the supernates removed for analysis (to measure the 

 extent of DNA depolymerization and loss). The supernates obtained from 

 DNAase-treated nuclei indicated a loss of more than 55 per cent of their DNA. 

 Parallel analyses showed that more than 70 per cent of the DNA in the treated 

 nuclei had been depolymerized and made soluble in cold 2 per cent HCIO4. To 

 test their capacity for ATP synthesis, the nuclear residues were resuspended in 

 buffered sucrose containing citrate (to inhibit further DNAase action) and were 

 shaken in air for 30 minutes at 38°. Following this second incubation, the nuclei 

 were collected by centrifugation. They were extracted with cold 2 per cent HCIO4 

 (to remove ATP and other acid-soluble nucleotides) and the extracts were neutral- 

 ized with KOH. The filtered extracts were then placed on Dowex-i (formate) 

 columns and the nucleotides were chromatographically separated and analyzed, 

 following the procedure of Hurlbert et aL(iS). The results are summarized in 

 figure 5/4 and B and in table 6. Nuclei treated with DNAase in this way contain 

 less than 30 per cent of the ATP found in untreated 'controls.' To test the possi- 

 bility that DNAase-treated nuclei can synthesize ATP but cannot retain it, the 

 supernates obtained by centrifuging the nuclei after incubation were examined 

 chromatographically; only traces of ATP were found. Since the ATP is not in 

 the nuclei and not in the supernate, it follows that removal of the DNA from 

 the nucleus impairs its capacity for ATP synthesis. 



A second type of experiment shows that the capacity for ATP synthesis is re- 

 stored to DNAase-treated nuclei if they are given a DNA supplement. The pro- 

 cedure used was similar to that described above. Nuclei were pretreated with 

 DNAase and centrifuged down, discarding the supernates. Some of the nuclei 

 were then resuspended in sucrose-citrate solutions containing thymus DNA; other 

 nuclei received no DNA supplement. The suspensions were incubated as before 

 and the nuclei were collected by centrifugation. The acid-soluble nucleotides were 

 extracted in cold 2 per cent HCIO4 and separated chromatographically. The 



