FREE NITROGEN COMPOUNDS IN PLANTS 687 
admit to the difficulty of distinguishing what is commonly called “incorporation” 
from what would be more strictly termed “net synthesis”. PROSSER ef al.°° experi- 
mented upon a pellet of material which was centrifuged after the separation of 
microsomes from rat-liver preparations. They were able to show that {!4C|leucine 
could be incorporated in the pellets, by an ATP-dependent mechanism, even though 
ribonuclease was present. In this way they demonstrated what they regard as 
ribonuclease-insensitive amino acid incorporation. By contrast, the microsomes and 
the supernatant sap lost the power of incorporating the leucine into protein when 
in the presence of ribonuclease, and this demonstrates what is regarded as ribonu- 
clease-sensitive amino acid incorporation. There is, therefore, the suggestion that 
certain animal cells may not incorporate all the amino acid into their protein by the 
now conventional RNA-activation route. (It is interesting to note in this connection 
that HENDLER™ (see also this Symposium) has described amino acid—lipid complexes 
which may also have some role in protein synthesis. ) 
Whereas recent well-known biochemical work (for summary see ref. 76) regards 
amino acid~RNA complexes as the immediate precursor of protein, there is other 
evidence that bears a somewhat different interpretation. RrE1H** supplied cells of 
ascites tumors with “C-labeled aspartic acid and was then able to isolate from the 
cells a radioactive complex which hydrolyzed to !C-labeled aspartic acid and un- 
labeled adenylic acid or uridylic acid, as the case may be. This work, therefore, 
supports the idea that such complexes could also function as donors of amino acids 
at the site of synthesis. Furthermore, it is not even universally accepted that in- 
dividual amino acids are invariably added singly into the orderly sequence in the 
chain which composes the protein molecule. ANFINSEN*® 6 has supplied radio- 
actively labeled amino acids to systems (hen oviduct or calf pancreas) which syn- 
thesize ovalbumin, ribonuclease and insulin, and has then hydrolyzed the resultant 
protein to recover the labeled amino acids from the different parts of the protein 
chain. ANFINSEN observed that a given amino acid may be differently labeled if it 
is isolated from different parts of the protein chain. For example, differences of up 
to 400% in the specific activity of alanine were observed. It is therefore inferred 
that single amino acids are not necessarily incorporated into the chain but that 
larger preformed units may be incorporated. However, using the rabbit, Simpson*? 
concluded that the individual amino acids were incorporated singly into the enzym- 
atically active protein that he studied (aldolase and glyceraldehyde-3-phospho- 
dehydrogenase). Using various labeled amino acids, he found that the specific 
activities of the different amino acids and their relations to each other remained 
constant throughout the whole of the molecule. This evidence is held to be consistent 
with the incorporation of individual amino acids rather than of preformed peptides. 
Different types of amino acid incorporating systems also seem to be required in 
higher plants, as indicated below. 
WEBSTER has followed a general pattern of investigation which was suggested by 
the work on animal systems and on microorganisms and has sought from higher 
plants evidence comparable to that which is produced by HOAGLAND and others 
(WEBSTER“!). One may now summarize some of WEBSTER’s latest findings. 
Using preparations from pea seedlings (ribonucleoprotein particles), WEBSTER has 
followed the synthesis of a “soluble” protein which has ATPase activity. This is 
followed by measuring the release of phosphate from ATP. The evidence of synthesis 
References p. 692/693 
