DISCUSSION 739 
compounds in the form of the soluble RNA and the AMP-amino acid can transfer their amino 
acid into TCA precipitates of a non-described character, and that the amino acid in that precipitate 
is held by bonds having the strength of peptide bonds. But this does not show that protein is 
being made; it does not show that the amino acid is found in any normal protein that this tissue 
can make. There are other arguments, for example, that equally uncharacterized systems for 
incorporation of amino acid into protein have been described which incorporate without the 
presence of amino acid activating enzymes or the presence of soluble RNA. Also in studies using 
intact cells that are making their normal proteins, the kinetics of passage of amino acid through 
the nucleic acid fraction is much too slow, or appears much too slow, to account for the total 
rate of incorporation into protein. On the other hand, this argument also is not conclusive, 
because the possibility exists that the RNA examined, which shows very little activity, may 
contain a small component which is turning over very rapidly. 
While I agree that there is no good evidence for peptide intermediates, I want to point out 
that there is also no good evidence saying that they do not exist. I think it is still valid for us to 
ask, are there stages less complex than the completed protein, perhaps less complex than partial 
protein on a template? I think that experimentally we are still not able to give an answer to 
this question, and we have to keep our minds open to this possibility. 
LortFIeLpD: There are a couple of things that I had not actually said. The last point is the only 
one I can remember and that is the question of the kinetics. Since it is not my work at all, I can 
defend it quite objectively. Frangois Gros has studied the kinetics of incorporation of valine 
and tyrosine, I think, in a bacterial preparation and has shown that some go per cent of the 
recorded rate of protein synthesis can be accounted for by the rise and subsequent fall in the 
amount of tyrosine and valine attached to the soluble RNA. Similar work has been done in ascites 
cells by LIrTLEFIELD and by HoaGLanpD, so that I do not think that this is a good argument 
against the system. 
But my point here was not to defend the generally accepted scheme for protein biosynthesis. 
I very pointedly said that I did not want to use that scheme to defend my proposition that free 
peptides are probably not intermediates in protein biosynthesis. I wanted to argue as far as 
possible on the basis that the experiments are faulty. I also said that there was not any incontro- 
vertible proof that peptides are not intermediates in protein synthesis. 
Leany: I would like to add a few remarks in regard to Dr. HENDLER’s comment concerning the 
isolation of an identifiable protein using the soluble RNA ribosome system. In the hemoglobin 
system studied by SCHWEET, reticulocytes produce 85 per cent or more of just one protein, hemo- 
globin. A protein labeled with radioactive amino acids and released from microsomes 717 vitvo 
has the same behavior as hemoglobin on columns and in its ammonium sulfate fractionation. The 
ratios of different amino acids in the isolated product are the same as the known ratios of those 
amino acids in hemoglobin, rather than the ratios of the amino acid concentrations in the in- 
cubation mixture. The per cent of N-terminal “C valine is the same as that found in hemoglobin 
from intact cells. 
ANDERSON: I would like to speak for just a moment to this point of what peptides might be 
doing. It is rather easy to isolate them from microsomes, for example, and ALBRIGHT and I 
showed some time ago that they turn over very rapidly. In a system where the machinery is 
breaking down at a very substantial rate, as is the case with RNA, for example, I do not believe 
that the cell is such a perfect system that it never makes any mistakes. It is not improbable that 
in a high percentage of instances when synthesis of a protein is started some mistake is made, 
and the pieces are subsequently broken down and reused, so that it may well be that the peptides 
which we isolate are merely mistakes. 
GRIFFITH: It has been adequately demonstrated by Professor W. C. Rosr and by others that 
an amount of total nitrogen which will maintain nitrogen balance if fed as protein may fail to 
do so if fed as an equivalent mixture of free amino acids unless additional calories are supplied. 
This observation implies that a mixture of free amino acids is not absorbed and used as efficiently 
in the body as is the mixture of the same amino acids entering the gastrointestinal tract in the 
form of protein. Some offer the explanation that small amounts of certain peptides are normal 
residual products of digestion, are absorbed as such and serve as particularly useful building 
stones in protein synthesis. Will the speaker comment on this possibility, please? 
LortFIELD: I think that there is one very likely answer which is that amino acids do not get 
through cell walls or through cell membranes very easily. We did an experiment in which we 
used radioactive albumin labeled in vivo, and compared the rate at which this and intravenously 
injected leucine entered rat liver cells. The albumin was taken into the cells about seven times 
more rapidly than the free leucine. Given the same amounts of radioactivity, the same specific 
activities of leucine in albumin or in free leucine, the specific acitivity of free intracellular leucine 
was seven times higher with albumin than with free leucine. I think that this probably corresponds 
to the observations that Dr. EaGLE has on glutamic acid. Does it get in very well? 
EaGLe: This may be the explanation, but on the basis of our experience with cultured cells it 
