740 Chairman: H. EAGLE 
would not be, because in cultured cells amino acids get into cells much more readily than does 
intact protein. As a matter of fact, we have had great dfficulty in demonstrating, at least to our 
own satisfaction, that small peptides get in as such. Our results are consistent with the idea that 
peptides are either hydrolyzed extracellularly by extracellular peptidase, or on the surface of 
the cell, with only occasionally an indication that the small peptides are taken in as such. The 
situation in an intact animal may be very different. 
SCHREIER: There may be another answer to the question of Dr. GrirritH. We know that if you 
feed monosaccharides to an animal, part of it is excreted in the urine, and much more is meta- 
bolized than if you feed polysaccharides. The same thing, of course, is true for proteins. The 
liberation time during digestion apparently has a great influence on the incorporation of the sub- 
stances into the cells. The human body, or any animals’ body, is not used to having high peaks. 
It wants to have very slow increases of any substances in blood because it is much easier for it to 
synthesize all the structural substances. In other words, much of the substances are lost by 
oxidation and other side reactions if they are given in a free uncombined from and not as poly- 
peptides, polysaccharides, and so on. 
Borsooxk: I would like to ask the speakers what they think now as to whether or not changes 
in the specific activity in the pool might or might not account for the claims of varying specific 
activity in proteins into which amino acids have been incorporated. This, I am aware, is a separate 
question from whether peptides or amino acids are incorporated, but it is conceivable that at 
some intermediate state, regardless of how it was arrived at, there might be exchange, and I 
would welcome any comments the speakers would care to make to this question. 
LoFTFIELD: You have specifically in mind an experiment like Kruu’s with labeled hemo- 
globins? I think that most of these experiments suffer from their experimental conditions, the 
rather low incorporation, the rather extended times, and the question of whether there was 
something going on besides protein synthesis. KRruuH, for instance, gets a hemoglobin isolated 
after some hours of im vivo synthesis and finds that on partial hydrolysis the radioactive amino 
acid is not released at the same rate as the #C-amino acid. Presumably this must involve, the 
formation of something besides a peptide bond or something besides the accepted scheme of 
synthesis. I know of no simple interpretation of the results. 
L. Miter: Why couldn’t you interpret the result of this experiment in terms of the difference 
in specific activity of the precursor in the pool at the time when the hemoglobin was being pro- 
duced? Was it clearly demonstrated that the precursor’s specific activity at the site of the hemo- 
globin synthesis was the same or different? 
LoFTFIELD: No. The actual experiment, as I recall, was between just glycine or phenylalanine 
injected into a rabbit. 
L. MILLER: You know that the red cells are formed in the bone marrow, and that the circulation 
of different parts of the bone marrow is not the same at different times, so that the pool, in one 
part of the bone marrow is not going to be the same at any particular time as it is at another. 
You are going to get all kinds of things there, and I do not see how it could be used to support 
one argument or the other. 
Borsook: I think I may add a couple of facts to this discussion. I know how KrRuu’s experi- 
ments were done, because they were begun in my laboratory. Some of them were done with 
reticulocytes which were suspended in a large volume of reaction medium. We know now that 
equilibration in terms of specific activity is obtained between the reticulocytes and the external me- 
dium in a very short time, a matter of a minute or two, so that in this case, I do not believe va- 
riations in specific activity can account for the result. My own question is on the analysis of the 
subsequently synthesized protein. I have always been puzzled that there should be a progressive 
change of specific activity according to the time at which the amino acid was released from the 
protein. This seems too orderly, somehow, for this kind of thing, and I had hoped tHat you 
might have some information to add to this. 
LortTFIELD: No, I haven't, although I would certainly think it appropriate for KRuH now to 
repeat this experiment and to isolate the peptides, a procedure which has become fairly routine. 
It remains, I think, the clearest example, or the most unchallenged example, of unequal labeling. 
ARONOFF: Professor Borsook, is there any possibility that there may be an isotopic fractiona- 
tion? If this were to occur in a series of consecutive steps it could result in extremely large differ- 
ences inspecific activity. 
Borsooxk: I do not know. 
Hatvorson: I would like to ask Dr. LoFTFIELD a question on his system involving ferritin forma- 
tion. As I understand it, you have a 5-minute half life in this experiment. How does that agree 
with the over-all protein synthesis of these cells? Is this a delay in the release, or are you looking 
at the heterogeneity in the completion of polypeptides? 
LoFTFIELD: In these particular experiments we had induced the synthesis of ferritin, so that 
by prior experiment we knew exactly the rate at which ferritin was being made, that is, the 
absolute rate of synthesis. Then, at zero time we injected C-valine or “C-leucine. During the 
