DISCUSSION 157 
the conditions of extraction. Although your primary extraction is made at a very low temperature, 
some of the subsequent steps are carried out at higher temperatures, and these are the ones that 
yield your peptides. Perhaps the greatest danger occurs in the finite interval that must occur in 
passing from —20° to go”. 
MircHELL: The material at — 20° is plunged directly into water that is boiling. That is the best 
I can do. We have done extractions at a little below zero, and successively at different tempera- 
tures, and we have obtained different increments and different sizes of products at these different 
temperatures. On the average, the size of what is extracted is larger as the temperature is in- 
creased. 
E. Roserts: Is there any possibility that these peptides are produced by methanolysis of bigger 
units? 
MitcHeE-t: I think that it would be necessary to have acyl amino acids or acyl peptides in order 
to get such reactions. You surely would not get it just from a peptide bond, and in any case 
hydrolysis would occur more rapidly than methanolysis in the 50 per cent solution. 
EaG LE: I have not so much a question as a comparison. In cultured animal cells, using cold 
TCA (trichloroacetic acid) to extract the amino acid pool, we are not plagued by this large 
amount of peptides. The TCA extract represents about 10 per cent of the total cell nitrogen. Of 
that, about half is in free amino acids, and the other half in peptides which release amino acids 
on hydrolysis. I rather suspect this is a difference in biological material, rather than in method 
of preparation. 
L. Mitrer: I presume that the larvae have a functional gastro-intestinal tract and I wonder, 
therefore, how you eliminate this gastro-intestinal contribution to the peptide “gemisch” ? 
MITCHELL: You can take the tract out. We have done this previously with small samples and 
the amounts of amino acids and peptides formed in the tract is negligible. 
E. Roserts: I do not think we have ever succeeded with paper chromatographic methods in 
finding any real evidence for occurrence of considerable amounts of peptides in brain extracts. 
Dr. TALLAN has used the column-chromatographic methods extensively in brain extracts and I 
wonder if he has found peptidic material in his extracts to any large extent. 
TaALLANn: We have never hydrolyzed the effluent from a chromatogram of brain, in the manner 
that Dr. MitcHE Lt described. However, when we hydrolyzed various portions of the effluent from 
Dowex-50 chromatograms of human urine, we did find a liberation of amino acids; glycine and 
glutamic acid were present in the largest amounts, but we also found aspartic acid, threonine, 
alanine, valine, isoleucine and leucine. 
CHRISTENSEN: With regards to Dr. TALLAN’s comment about bound glycine in the urine, this 
appears largely to be hippuric acid, which we have also been able to detect in the liver. In general, 
we must always be suspicious that amino acid conjugates found in tissue extracts or body fluids 
may not be peptides at all. 
L. Miter: That brought to mind another perhaps totally extraneous observation; that is, in 
working with dogs years ago, we found that we could get enormous increases in urinary peptides 
by infusing intravenously enzymatic digests of protein. Are these Dvosophila larvae being fed a 
completely defined fodder or are they being fed a partially digested undefined diet containing 
peptides which may not be particularly appealing to them and which are consequently accumulat- 
ing somewhere in the back-waters, if you will, of the extracellular fluid? 
MITCHELL: Our larvae are fed whole, live yeast. 
L. MILLER: And whole live yeast have no peptides at all that are comparable to those you find 
in the larvae. 
MitTcHELL: In the amount present, no. Whole live yeast contain peptides, but the amounts 
present in the intestinal tract are negligible. 
L. MILLER: Of course, one would assume that in the process of transforming whole live yeast to 
whole live larvae there are no peptides formed excepting those that the beasts had a fancy for; 
that is, metabolically speaking? 
MircHeELL: Well, only the larvae could answer that. 
Hatvorson: I have had some experience with protein breakdown in starving yeast cells and 
I would like to have one point clarified. If you look at non-starving larvae, do you see any differ- 
ence in the peptide content as compared with those that you place under starvation conditions? 
CHEN: There is some increase of peptides, but not those originally present. 
