170 Discussion 



mc to a 270-g. rat), in the hope that we would get a high level of radio- 

 activity incorporated into protein in a few minutes (cf. Fig. 3, Little- 

 field et ah, 1955, loc. cit.). 



Work: We may have missed a peak which you picked up in using a 

 higher dose. We used about 35 mc/kg. animal. Ours was a comparable 

 specific activity, but a much smaller dose. 



Zamecnik: As regards the two different ribonucleoprotein fractions, in 

 the case of the rat liver we obtained particles such as this using sodium 

 deoxycholate fractionation. Ultracentrifugally, the ribonucleoprotein 

 fraction behaves as one peak, although I don't think that necessarily 

 implies very much. But when my colleagues Littlefield and Keller pre- 

 pared ribonucleoprotein particles from the Ehrlich mouse ascites tumour 

 they found three closely parallel ribonucleoprotein peaks. I don't 

 know whether the specific activity in those were all the same. 



Work: We also found that if we fractionated the supernatant with 

 ammonium sulphate we got very considerable variation in activity. 



Zamecnik: We have carried out some fractionation of this 100,000 g 

 supernatant protein too. Dr. Hoagland has rather good evidence that 

 there is not just one enzyme or one enzymatic site involved in activation 

 of all amino acids. I don't know whether there is one enzyme for each 

 amino acid, but there is evidence for three or four separate ones so far. 

 De Moss and Novelli (1955, Biochim. biophys. acta, 18, 592) have con- 

 firmed this finding in bacteria, and they found evidence for activation of 

 8 separate amino acids, and think there is more than one enzyme 

 involved. 



Work: That would fit in with our experience. Whenever we fractionate 

 we divide it into a lot of fractions with less activity than the original. 



Zamecnik: We have not had good luck in fractionating this pH 5 

 enzyme and then using these fi-actions separately for the whole incorpor- 

 ation process. We seem to lose the activity on fractionation, and my 

 impression is that we have several enzymes involved, which are going 

 into different fractions. 



Popjak: Is it not possible that the aminoacyl nucleotide or amino- 

 acyl AMP is only a first intermediary so to speak, and that the hydrox- 

 amate that you get in these preparations perhaps really comes from 

 another type of activated amino acid ; in the acetate activation reaction 

 it is also postulated that it is the acetyl AMP which is the first inter- 

 mediate and then acetyl coenzyme A is formed. I think that this is very 

 likely, particularly in view of what Dr. Work has said about the instabil- 

 ity of mixed anhydrides of amino acids with AMP. 



Zamecnik: That fits in with the idea that maybe one has an enzyme- 

 bound activated intermediate. 



Work : One point I should mention : we found that the rate of appear- 

 ance of ATP is very much faster than the rate of appearance of hydrox- 

 amate. Does that agree with your findings? 



Zamecnik: Yes, our preparations contain our "ATPase", unrelated 

 to amino acid activation, in addition to the ATP splitting involved in 

 this process. 



Popjak: It is very likely that you have more than one activating 



