NUCLEAR PROTEIN SYNTHESIS 279 



Discussion 



Canellakis : Have you tried adding ribonucleic acid instead of deoxyribonu- 

 cleic acid to your isolated ribosomes ? 



Allfrey: Yes. In isolated ribosomes the addition of commercially prepared 

 yeast RNA failed to stimulate the uptake of [i-'*C]-leucine. 



Canellakis : Was it a high molecular weight polymer ? 



Allfrey: Yes. That is, it was non-dialyzable. 



Arnon : When you speak of ATP synthesis by the nucleus do you imply that 

 the nucleus is self-sufficient with respect to its ATP requirement for protein syn- 

 thesis or does it still depend on ATP formation by mitochondria ? 



Allfrey : I would say that for amino acid incorporation in isolated thymus 

 nuclei it is not necessary to add mitochondria or some other ATP-generating system. 

 Amino acid uptake into proteins by isolated nuclei is not as active as in whole 

 cells (perhaps only one-third as active) but the process can continue actively for 

 8-9 hr. in the absence of mitochondria. Moreover, mitochondrial contamination is 

 not a problem because we inhibit mitochondrial phosphorylation by adding Ca-+ 

 ions. The nuclei are still able to carry out all of these synthetic reactions. 



Arnon : What is the substrate for ATP formation ? Is oxygen being consumed ? 



Allfrey: Yes, oxygen is consumed. The QCOg) usually lies between i and 2. 



Arnon : But what is the chemical substrate ? 



Allfrey: We don't know how the coupling of oxidation and ATP formation 

 takes place. We are working on that, and know that glucose is metabolized in these 

 nuclei. 



ScHOFFENiELS : Did you find competition between the l and the d forms of the 

 same amino acid in the process of transfer from the solution to the nuclei ? 



Allfrey: There is no competition between the l and the d forms. It is only the 

 L form of the non-radioactive amino acid which will compete with the radioactive 

 amino acid ; the d form does not. 



Schoffeniels : If you add both, will not the d form be inhibitory ? 



Allfrey: It has no action at all at the concentrations we have tested. 



Herbert : I wonder if you have characterized the RNA, which I presume is 

 much like soluble RNA, further than to establish that the amino acid complexes 

 with adenylic acid, and whether you have characterized the activating enzymes to 

 any extent. It has been reported that activating enzymes isolated from nuclei are 

 different from the cytoplasmic enzymes with respect to certain amino acids. 



Allfrey : We have tried both specificity reactions, testing the nuclear amino 

 acid-activating enzymes and cytoplasmic enzymes from guinea-pig liver, and 

 ribonucleic acids from diflferent types of nuclei. Now, the answer you get depends 

 on the amino acid you use. If you use leucine, both Dr. Webster and I agree there 

 is no sign of specificity, i.e. the nuclear enzyme will transfer to cytoplasmic RNA 

 or the RNA of other nuclear types, and the cytoplasmic enzyme will transfer to 

 nuclear RNA. But in Dr. Webster's experiments with alanine-activating enzymes 

 there is evidence of specificity between liver nuclear enzymes and cytoplasmic 

 enzymes. We haven't checked this with thymus nuclei. We have now started to 

 characterize the amino acid " carrier "-RNA of the thymus nucleus. We do not 

 call it " soluble "-RN A because in our system it occurs in association with the 



