VIII. PROTEIN SYNTHESIS AND GENE ACTION 373 



(Nathans and Lipmann, 1961). The partially purified E. coli enzyme 

 fraction already referred to was completely inactive in the transfer 

 of amino acids from amino acyl-RNA to reticulocyte ribosomes. Sim- 

 ilarly, rabbit reticulocyte pH 5 supernatant was inactive in the E. coli 

 system. The significance of this type of specificity is not understood. 



B. FORMATION OF THE PEPTIDE CHAIN 



Understanding of the mechanism of assembly of the amino acids may 

 be expected to yield information on the central problem of how amino 

 acid sequence is specified. One approach has been to study the distribu- 

 tion of a C^^-amino acid in various portions of a specific protein synthe- 

 sized in the presence of this labeled amino acid. If the specific activity 

 of the C"-amino acid is different in various parts of the peptide chain 

 (unequal labeling) , information about intermediate pools or sequence of 

 peptide bond formation can be deduced (Loftfield, 1960). 



1. Labeling Patterns in Intact Cells 



Unequal labeling of specific proteins has been observed in a number 

 of systems. The clearest example, perhaps, is the unequal labeling of the 

 ovalbumin synthesized in chicken oviduct (reviewed by Steinberg et al., 

 1956). Unequal labeling was clearly demonstrated by analysis of the 

 products of (a) partial acid hydrolysis, (6) peptic digestion, and (c) 

 sequence analysis of the hexapeptide liberated by subtilisin. The data 

 were interpreted to indicate the existence of intermediate pools of some 

 kind, or an ordered synthesis of the chain. It is not possible to distinguish 

 between these possibilities as the author noted. Equal labeling of goat 

 lactalbumin was observed by Askonas et al. (1955). This is consistent 

 with any of the hypotheses discussed above, since the time to synthesize 

 a lactalbumin molecule was not determined. 



The a-amylase secreted by late log phase B. siibtilis is the subject 

 of a careful study by Yoshida and Tobita (1960). C^*-leucine was added 

 to the culture during the later stages of growth, a-amylase was isolated, 

 and leucine was isolated from positions close to the C-teiTninus, the 

 N-terminus, and from eight tryptic peptides. The highest specific activity 

 leucine was found close to the C-terminus, and the lowest close to the 

 N-terminus. Furthermore, the specific activities of the leucine in the 

 eight tryptic peptides fell between these limits. This experiment, then, is 

 suggestive of an ordered synthesis of the polypeptide chain of ^-amylase, 

 starting at the N- terminal end, and finishing at the C-terminal end. It 

 should be noted, however, that a high molecular weight precursor of 

 amylase was present before the isotope experiment began and the results 

 may mean that this precursor was completed. 



