Discussion 171 



enzyme, because even in the case of simple homologues like fatty acids 

 several activating enzymes are known, all of which have different chain- 

 length specificity; so that in a group of more diverse substances like 

 amino acids it is even more likely that you should have more than one 

 activating enzj^me. 



Bracket: Do you know (a) whether the protein present in these very 

 small particles has any of the enzymatic activity which is usually 

 associated with microsomes; (b) whether it is something like a histone 

 or any of the basic proteins ; and (c) whether removal of the ribonucleic 

 acid from these very small granules has an inhibitory effect on incorpora- 

 tion? 



Zamecnik: We have added ribonuclease in very small concentrations, 

 about 1 yLg./mh, and have found it inhibits the incorporation reaction 

 completely. There is some breakdown of RNA, but the inhibition 

 appears to exceed it enormously. We have done some preliminary frac- 

 tionation experiments on these particles. If we add ribonuclease at a 

 concentration of 5 or 10 [ig. /ml. and incubate it with these particles for 

 10-15 minutes, everything comes down in a coagulum, all stuck together. 

 We were disappointed with that experiment. But we have also incubated 

 ribonucleoprotein particles with between two-tenths and 1 y.g. of 

 ribonuclease in a cold room at 4°C for 3 days, in the presence of a fairly 

 high salt concentration; under those circumstances the ribonucleo- 

 protein broke down into several fragments. A fraction which did not 

 centrifuge down at 100,000 g for 2 hours had about twice the specific 

 radioactivity of the fraction which did spin down. This soluble protein 

 fraction was placed in the electrophoresis machine (we have only done 

 this once). There were three components. We cut between the slowest 

 moving peak and the other two peaks and found that the slowest 

 moving peak had half the specific activity of the others. That is a crude 

 fractionation, but it suggests that we may be able to break this ribo- 

 nucleoprotein down by some procedure. I have no doubt that it consists 

 of more than one protein. We suspect that only a small portion of the 

 protein components ds involved in the rapid synthetic mechanism. 



Holmes: I should like to know if Dr. Zamecnik has any evidence of the 

 RNA itself being broken down and rebuilt during amino acid incorpora- 

 tion. 



Some years ago we prepared a crude cytoplasmic ribonucleoprotein 

 fraction from the tumours of rats injected during life with ^ss-methio- 

 nine and ^sp. This fraction was prepared by precipitation at pH 5-0 

 after removal of the nuclei and contained, all the remaining RNA and a 

 considerable amount of protein. The uptake of ^^p into the RNA 

 seemed to parallel the incorporation of methionine into the protein. 

 X-ray irradiation of the tumour in vivo had no effect upon the uptake 

 of either of the labels, whereas the injection of shock-producing chemicals 

 caused a proportionate reduction in the uptake of both. 



Zamecnik: In Potter's laboratory experiments were performed on in 

 vitro incorporation of labelled orotic acid into RNA in rat liver homogen- 

 ates; they used almost the same components that we have used here, 

 and found that the orotic acid does make its way into the RNA molecule. 



