I06 NITROGEN METABOLISM 



The mechanisms operative in the formation of peptide 

 bonds are only one aspect of protein synthesis; it is also 

 necessary to consider (i) the means whereby the correct 

 sequence of amino-acids is attained in a peptide chain, (ii) 

 if the protein molecule comprises more than one peptide 

 chain, how such chains are linked together and (iii) the 

 spatial arrangement of the amino-acids and the peptide 

 chains. Major advances in solving these problems await the 

 determination of the structure of specific proteins (cf. 

 Sanger's recent elucidation of the amino-acid sequence in 

 the peptide chains of insulin). Investigations of protein syn- 

 thesis in animal tissues have been mostly confined to an 

 examination of the conditions in which isotopically labelled 

 amino-acids are incorporated into material precipitated by 

 trichloracetic acid, i.e. presumably bound in proteins or 

 polypeptides. Such experiments have proved little except 

 that incorporation is associated with the utilization of meta- 

 bolic energy, and great care is required in making deduc- 

 tions from the observed results [49]. A more direct approach 

 is provided by studies of the synthesis of a specific protein, 

 e.g. an enzyme whose activity can be estimated and used as 

 an index of concentration. Evidence has gradually accumu- 

 lated to the effect that at least in certain cases the adaptive 

 formation of enzymes is the outcome of de novo protein syn- 

 thesis rather than the mere subtle modification of existing 

 proteins, i.e. enzymes or 'enzyme precursors' [30]. If this is 

 true, then the formation of adaptive enzymes would appear 

 to offer a most promising field for studying protein synthesis 

 in micro-organisms. Concentrations of 2:4-dinitrophenol 

 and azide which, although not affecting respiration and the 

 fermentation of carbohydrates, inhibit the uptake of inor- 

 ganic phosphate and in consequence the synthesis of energy- 

 rich phosphate bonds, also inhibit adaptive enzyme forma- 

 tion and the incorporation of isotopically labelled amino- 

 acids into peptides and proteins. 



Certain observations indicate that the assembling of the 

 constituent amino-acids is a preliminary step in the synthesis 

 of a protein. For example, the synthesis of the adaptive 

 enzyme nitratase in washed cells of Esch. coli [33] and of 



