28o VINCENT G. ALLFREY 



nuclear ribosomes. This may be because we flood the system with Ca'^^ ions during 

 the course of isolating the nuclei. 



Bergeron : Would it be correct to say that DNA-ase has free access by diffusion 

 into a compartment into which alanine does not diffuse freely but has to be moved 

 by active transport ? 



Allfrey : I think that is right. It is not only true for DNA-ase, which has a 

 molecular weight of 6i coo, and for RNA-ase with a molecular weight of about 

 13 000, but also for some of the polyelectrolytes which we have used with molecular 

 weights up to 200 000. However, an alternative explanation may be offered to the 

 compartment hypothesis. Perhaps the apparent presence of alanine in a free "pool" 

 is an instance of specific adsorption rather than membrane-limited compart- 

 mentalization. 



Bergeron : Might it not be true that you have different pools in the one system 

 you are dealing with, which you call the nucleus? 



Allfrey : I think it's true. Certainly the effect of sodium in stimulating amino 

 acid entry into the "pool" differs greatly for different amino acids. 



Porter : Did you imply that the radioactive sodium is concentrated intra- 

 nuclearly or in the perinuclear space ? This arrangement of channels bringing it to 

 the perinuclear space would account for the increased density in the nuclear area 

 in a radioautograph of a whole cell. 



Allfrey : The figure shown was a radioautograph of a section of the frog 

 oocyte. The high grain density over the nuclear area indicates the presence of 

 ^^Na within the nucleus. This result has been checked by direct isolation of these 

 nuclei. Dr. H. Naora made a set of very small chopsticks out of wood and devised 

 techniques for removing a single nucleus from frozen oocytes. Thus we were able 

 to count the nuclei separately from the cytoplasm. 



Porter: It seemed to me that sodium ought really to be concentrated in the 

 perinuclear space and that the migration of the sodium is through the pores in the 

 nuclear envelope from the cytoplasm matrix. 



Allfrey : Well the sodium is freely diffusible in our system of isolated thymus 

 nuclei. 



Mitchell: Could you tell us what evidence there is that the alanine which is 

 accumulated is free inside the nucleus ? This is a difficult problem, and one that 

 many people have encountered in trying to prove that a given process was active 

 transport. How do you get the alanine out to show that it was free ? 



Allfrey : To put it as briefly as possible, we extract with cold trichloroacetic or 

 preferably cold 2% perchloric acid. This leaves the proteins behind. Then we take 

 the perchloric acid extract which is concentrated and chromatographed. We find 

 a single spot corresponding in Rf to free alanine. Now this does not mean some 

 other intermediate wasn't formed in the process, but we have not yet found evi- 

 dence for any other radioactive product. 



