Discussion 169 



DISCUSSION 



Work: We too have been following uptake of radioactive amino acids 

 into liver preparations in the intact animal, and have adopted a similar 

 centrifugal fractionation scheme followed by a scheme of salt fraction- 

 ation of the guinea pig liver microsomes. We found also that we had 

 peak activity in the microsomes. A nucleoprotein fraction obtained by 

 salt fractionation of the microsome material showed a quite clearly 

 defined peak thirty minutes after injection; we could separate out two 

 nucleoprotein fractions, one of which showed this peak of activity and 

 one which did not. Why there should be a peak at thirty minutes in our 

 guinea pigs and a peak at a very much shorter period in Dr. Zamecnik's 

 rats, I don't know. It looks as though we are both handling very 

 similar types of material. 



On the additional steps in the reaction, the activation of the carboxyl 

 group, we have been able to confirm Hoagland's work. Hoagland very 

 kindly wrote and gave us details before he published this. 



I was interested in your remarks about the use of leucine AMP 

 anhydride ; we have made alanine AMP anhydride, and have found that 

 it is unstable ; in aqueous solution it polymerized rapidly to its peptide. 

 I would be interested in hearing if anybody had managed to get a suf- 

 ficiently stable preparation to work with. It seems to me that the amino 

 group must be protected in some way or other in the enzymic carboxyl 

 activation process. As you say, it may be attached to the enzyme 

 surface in some way which protects the amino group. Another distinct 

 possibility, I think, which is worth consideration, is that the amino 

 group may also be protected by a phosphoramide linkage. We made 

 some phosphoramides and found that metabolically they were indis- 

 tinguishable from the free amino acids; in other words the phosphor- 

 amide group obviously comes off very quickly in a biological system, and 

 it seems to me that it is a possible intermediate which we ought to 

 consider. 



Another point that I think we ought to keep in mind is that these 

 mixed anhydrides are so unstable that if you add any acceptor which is 

 potentially capable of forming an anhydride group, even in the absence 

 of any enzyme, you get mixed anhydride. Thus if you take AMP and, 

 say, leucine phosphate anhydride you get ATP from it without any 

 enzyme. 



Zamecnik: De Moss, Genuth and Novelli have just published (1956, 

 Fed. Proc, 15, 241) their results, and thfeir yield of this leucyl AMP 

 anhydride was very poor, about 7 per cent. The half-life of this com- 

 pound was several minutes at pH 7, making possible a study of its con- 

 version to ATP by the enzyme in the presence of pyrophosphate. With 

 regard to your work on guinea pigs, the technical details of our experi- 

 ments with the rat and yours with the guinea pig may be a little dif- 

 ferent. I suppose we both injected intravenously? 

 Work: Yes. 



Zamecnik: In one type of experiment we gave a small dose of 

 ^*C-leucine of a very high specific activity, about 9 mc per m-mole (1 -4 



