164 Zamecnik et al. 



of whether this peptide bonding represents de novo synthesis 

 of a peptide chain or exchange of a single amino acid for its 

 non-radioactive isotope within the interior of an existing 

 peptide chain. To begin with, Littlefield and Keller (1956) have 

 shown that biologically active microsomes, labelled by incuba- 

 tion of ascites tumour cells with ^^C-leucine, do not lose their 

 protein label when incubated in a complete cell-free system 

 containing • 01 m ^^C-L-leucine. In another type of experi- 

 ment, liver microsomes, labelled by 3-minute cell-free incu- 

 bation with ^*C-leucine or ^^C- valine of high specific activity, 

 did not lose this radioactivity when ten times as much inert 

 leucine or valine was added (Littlefield et at., 1955) for a 

 further incubation period. These specific pieces of data 

 argue against a simple exchange reaction as the mechanism of 

 labelling. Reasoning along more general lines, the hydrogen 

 bonding and specific three-dimensional patternization of a 

 completed protein molecule would appear to prohibit exchange 

 of a single amino acid for another located in the interior of the 

 peptide chain. The data are more compatible, therefore, with 

 the conception that the labelled amino acids measure a small 

 amount of de novo synthesis of long chain peptides in the 

 ribonucleoprotein particulate fraction of this cell-free system. 

 During the 10-minute cell-free incubation at 37° in which the 

 labile ribonucleoprotein particle retains its biological activity, 

 up to 0-2 per cent labelling of the ribonucleoprotein leucine 

 occurs. 



There are five essential components of the cell-free incor- 

 poration system: (1) the microsome fraction, (2) the pH 5 

 precipitable enzyme fraction, (3) ATP (and usually an ATP- 

 regenerating system), (4) GTP or GDP, and (5) the labelled 

 amino acid. The general method of preparation of the 

 protein fractions is indicated in Fig. 1. If any one of these 

 constituents is omitted, the incorporation suffers (Keller and 

 Zamecnik, 1956). It has recently been possible (Littlefield 

 and Keller, 1956) to simplify this system a little by using 

 cellular fractions prepared by 0-5 M-NaCl extraction and 

 centrifugal fractionation of distilled water lysates of Ehrlich 



