37. NUCLEIC ACID AND PROTEIN SYNTHESIS 351 



of these cell-free systems. These matters have been discussed by Loft field 7 

 particularly. Ideally we should like to have a system which would bring 

 about a directly measurable net increase in protein. Some promising results 

 have recently been obtained along these lines which will be discussed later. 

 However, most cell-free systems thus far studied have failed to show a net 

 synthesis and workers in the field have been forced to measure incorpora- 

 tion of tagged amino acids (usually labeled carboxyl-C 14 ) into protein. In 

 the light of our current knowledge, the minimum criteria to be met in 

 equating such incorporation with protein synthesis may be said to be the 

 following: (1) the incorporation of an L-amino acid must be irreversible in 

 the sense that once the amino acid has entered protein the total amount 

 of C 14 -amino acid in protein in the system cannot subsequently be reduced 

 by a continued incubation in the presence of an excess of the same amino 

 acid, unlabeled. (2) The incorporation should be dependent upon the addi- 

 tion of a metabolic energy source, adenosine triphosphate (ATP) or an 

 ATP-generating system. (3) The incorporated amino acid must be shown 

 to be in true alpha peptide linkage in protein, as evidenced by its appearance 

 in identifiable peptides upon partial hydrolysis of the protein. (4) The 

 amino acid should be located within the peptide chain, not in terminal 

 positions. Other criteria which are desirable but which have not been con- 

 clusively demonstrated in all systems are: (5) that the amino acid appear 

 in a single, specific, isolatable protein of the cells of origin, and (6) that in- 

 corporation of one C 14 -amino acid be dependent upon the addition of the 

 other amino acids naturally found in protein. 



"Incorporation" refers to an irreversible, energy-dependent inclusion of 

 a monomer in appropriate covalent linkage in a polymer. "Exchange" is a 

 term frequently used in discussing the significance of amino acid incorpora- 

 tion work, describing a process by which a free monomer replaces an inter- 

 stitial monomer in a polymer chain without breakdown of the latter. An 

 interesting chemical system which will carry out the insertion of an amino 

 acid into a peptide chain has been developed by Brenner. 17 However, with 

 the exception of the special case of the transpeptidation reactions 18 (whose 

 role in amino acid incorporation reactions remains unclear), there is no 

 evidence that such a phenomenon occurs in relationship to protein-amino 

 acid interaction in biological systems. (The often quoted experiments of 

 Gale and Folkes, 19, 20 in which a reversible incorporation of a free amino 

 acid into protein appeared to occur, no longer supports the "exchange" 



17 M. Brenner, Ciba Foundation Symposium on Amino Acids and Peptides with Anti- 

 metabolic Activity p. 157 (1958). 



18 J. S. Fruton, Harvey Lectures 15, 64 (1957). 



19 E. F. Gale and J. P. Folkes, Biochem. J. 59, 661 (1955). 



20 E. F. Gale and J. P. Folkes, Biochem. J. 59, 675 (1955). 



