BIOCHEMICAL PROPERTIES OF THE ISOLATED NUCLEUS 205 



DISCUSSION 



J. W. Green, V. Alljrey, R. E. Beyer, L. Ernster, D. A. Marsland, E. L. Ktiff, 

 J. W. Littlefield, A. Marshak, W . S. Vincent 



Dr. Green: Do you have any information on the specific role sodium plays in the 

 nucleus? 



Dr. Allfrey: At present no specific chemical role is known for sodium ions in 

 nuclear metabolism. It is unlikely that it is involved only in amino acid incorporation, 

 because the uptake of adenosine-8-C" into nuclear RNA has also been found to be 

 sodium-dependent. It might be added that the thymus nucleus is not the only instance 

 of a nucleus displaying a sodium requirement. Several years ago Abelson and Duryee 

 (P. H. Abelson and W. R. Duryke, Biol. Bull. 96: 205, 1949) showed by radioauto- 

 graphic techniques that the nucleus in intact frog oocytes rapidly takes up Na-*C1 from 

 the medium. 



Dr. Beyer: In view of the synthesis of DPN by the nucleus, I wonder if you have 

 tried to add DPNH or other electron donors as well as AMP or ADP to see if you can 

 measure a greater synthesis of ATP.? 



Dr. Allfrf.y: In synthesizing ATP the nucleus will phosphorylate only the AMP 

 which it contains, and added AMP or ADP is not acted upon. We have not tried the 

 combination you suggest to see if DPNH will alter the rate of ATP synthesis. 



Dr. Ernster: Have you tried to measure oxygen consumption, or also to replace oxy- 

 gen by an artificial hydrogen acceptor, e.g. ferricyanide, reduction of which may be 

 easier to measure than is oxygen consumption? 



Dr. Allfrey: We have measured oxygen consumption in the isolated nuclei: it is 

 definite, but quite low, and we have not tried to calculate P/O ratios for this system. 

 The suggestion to use ferricyanide as an acceptor is a good one and should be tried, 

 but we are more interested at present in trying to unravel some of the problems of 

 nuclear protein and RNA synthesis. 



Dr. Marsland: After you remove the ATP by acidification and adding acetate, can 

 you restore it in active form? 



Dr. Allfrey: The addition of acetate (0.02 m) at pH 5.1 removes 85-90 per cent 

 of the nucleotides from the nucleus, including both AMP and ATP. It should be men- 

 tioned that, at these lower pHs, the nucleoside triphosphates and diphosphates are 

 largely con\erted to the monophosphate form, and that when they are removed, potas- 

 sium is released at the same time. Attempts to restore nucleotides to nuclei treated in 

 this way have not been carried out. 



Dr. Kuff: Is any incorporation actively demonstrable in preparations of nuclei that 

 have been disrupted by mechanical or other means? 



Dr. Allfrey: Nuclei which are broken by high speed stirring in a blender are 

 completely inactive as tested by amino acid incorporation. The same result is ob- 

 served in nuclei disrupted by freezing and thawing. But Drs. Breitman and Webster 

 (T. R. Breitman and G. C. Webster, Fed. Proc. 17: 194, 1958) have recently reported 

 that sonic disintegration, if carefully carried out, gives nuclear fragments which can 

 incorporate radioactive adenine and AMP into nucleic acids. 



Dr. Littlefield: Two quick cjuestions: First, is there any way, perhaps by differ- 



