730 R. B. LOFTFIELD 
by which the genetic information is used in the synthesis. The mechanism for the 
polymerization reaction is also largely unknown, although GTP is required and 
certain drugs can interfere with the process. 
How well does a peptide mechanism fit these observations? It is extremely difficult 
to understand how even 20 different amino acids can be distinguished from each 
other by the activating enzymes which must also select among twenty different 
S-RNA’s; it is nearly inconceivable that there should be a sufficient number of 
enzymes to distinguish among the thousands of potential peptide intermediates and 
the similar number of S-RNA’s that would be postulated for the peptides. In truth, 
since there is a limited amount of S-RNA present in the cytoplasm and since at 
least 90°, of it can be shown to react with one or another of the twenty natural 
amino acids there simply is not any significant amount of peptide specific S-RNA. 
It might be suggested, of course, that peptides are incorporated by a mechanism 
that does not involve S-RNA, for TUBoI AND HuziNo?’ have shown that peptides can 
be activated. However, both BEercG?® and we** have found that the specificity of 
amino acid-activating enzymes is not absolute, that other natural and unnatural 
amino acids may be activated but that the Michaelis constants and the transfer of 
the activated amino acids to S-RNA are such as to preclude the operation of an 
activating enzyme on any but its natural substrate. Although we have not examined 
Tupol’s peptides, I am certain that we will find that they are not activated to a 
significant extent under conditions of concentration and competition such as would 
exist in a cell. 
One might then say that perhaps the peptides are incorporated into protein by a 
route that doesn’t involve even the activating enzymes. Such systems have in fact 
been described®*. In such cases it may be that protein is actually not being synthesized, 
that the apparent synthesis is a manifestation of an artifact. However, at this point 
I think it would be wisest to reserve judgment. There are, after all, many syntheses 
of polypeptide or protein substances which clearly do not involve the reaction scheme 
presented above. Glutathione synthesis proceeds through a free peptide and the free 
amino acids are activated by a different mechanism**. The polypeptide material of 
cell walls involves not only free peptide intermediates but the coupling of one peptide 
to another®®. The “synthesis” of trypsin from trypsinogen is clearly not mediated 
by microsomes and activating enzymes. It might be surprising but surely not im- 
possible to find a protein whose final completion requires transpeptidation rather 
than hydrolysis. 
I would end on this note. I feel that there has been no persuasive evidence that 
free peptides are, in fact, intermediates in protein synthesis. There have been many 
conscientious efforts to discover evidence for such intermediates and in each case 
the evidence is negative. The question remains unsettled only because an argument 
cannot be proved conclusively by an accumulation of negative data. Further negative 
data will add little to the argument and the unlikelihood of getting positive evidence 
of peptide participation seems so small that I can think of several more promising 
areas of research. 
ACKNOWLEDGEMENTS 
The preparation of this material was supported by AEC Contract AT (30-1)2643 and 
U.S. Public Health Service Grant C-2387. 
References p. 737 
