DYNAMIC ASPECTS — AMINO ACID POOL TURNOVER 
NI 
e) 
[op 
DISCUSSION 
Chairman: JOHN REINER 
Roperts: I wonder if Dr. EAGLE has compared the pools in cells and medium when more complex 
materials were used in the environment, with those found in these pools when cells are suspended 
in the relatively purified culture media which contain only a small amount of protein. I ask this 
question because we found no similarity whatsoever, between the pools observed in ascites tu- 
mor cells grown in animals and the pools found in the ascitic fluid in which the tumor cells were 
suspended. However, if we separated these cells from the fluid and put them into various buffers 
the pools began to look quite similar. I was just wondering whether we might be altering relation - 
ships between what Dr. Cowl® has described as “internal” and “external” pools. Perhaps some 
factors in the ascitic fluid might shift more of the amino acids to the so called “internal” pool 
in mammalian cells while removing them into buffer might cause a shift to the “external” pool. 
Eac te: In the range of 0 to 20% serum, which means, therefore, in the range of o protein to 
approximately 1.5°, protein in the environment, there was no effect whatever on the steady 
state relationship between the cell and the medium. We have studiously avoided using salt 
solution as the medium first, because it is lethal to these cells, and rapidly so, and second, because 
the equilibration between cell pool and medium is so rapid. The only way we can get meaningful 
pools in these cells under these conditions is to chill monolayer cultures and wash just once, 
very quickly with cold salt solution. If one washes three or four times, one begins to lose amino 
acids from the pool. Parenthetically, we have found that amino acid pools obtained from suspen- 
sion cultures by centrifugating the cells preliminary to treatment with TCA result in artifact 
pools, due to cell proteolysis in the course of the centrifugation and packing. 
Hatvorson: All of the claims in microorganisms for exchange incorporation, have essentially 
been explained by the finding that one was dealing with cell wall synthesis. At the moment there 
is no convincing example of such exchange incorporation. Dr. EAGLE’s rather provocative observa- 
tions lead me to ask this question. We have always been impressed with the extent to which 
turnover takes place when you carry out long-term experiments, suggesting that the bulk of the 
protein is involved. Now, in the experiments you just mentioned at the end of your talk, were 
these carried out long enough to implicate a large fraction of the protein? Also, could you com- 
ment on the characterization of the newly incorporated material in the presence of the inhibitor? 
Eac te: If turnover is studied in a non-growth medium, in which protein synthesis is prevented 
quite simply by leaving out one or more essential amino acids from the environment, then, as 
Dr. HaLvorson indicated, in a good experiment the cells continue to incorporate for as long as 
72 hours, and at the same rate of 1 per cent per hour. One must conclude, therefore, that most 
of the cell proteins are involved in the turnover process. Since the turnover is observed with all 
the amino acids studied, essentially all of the amino acid residues of the cell protein are similarly 
involved. 
If metabolic inhibitors are used at effective concentrations, the cells die and slough off in less 
than 72 hours. In our best experiments, however, we have gone 24 to 48 hours, at a turnover rate 
of 0.5~-1 per cent per hour. The amino acids incorporated are in the hot TCA residue, and presum- 
ably in peptide linkage. We can only conclude that, at least to this extent, we are getting the 
continuing incorporation of labeled amino acids in peptide linkage in protein. I hold no brief for 
the thesis of exchange incorporation. I only say that it is consistent with our data, and I find it 
difficult to reconcile these data with the classic view of protein turnover as degradation and re- 
synthesis unless one assumes two quantitatively different kinds of protein synthesis. 
GurorFr: The isolated intact diaphragm and also the brain slice will maintain its normal 
endogenous concentration of amino acid when incubated for long periods of time in the pre- 
sence or absence of substrate or in the presence of metabolic inhibitors. However, it will rapidly 
lose additional amino acid which has been accumulated in vitro. I would like to know, Dr. EAGLE, 
if the tissue culture maintains a constant low level of amino acid while losing the newly accumu- 
lated pool? 
EaGte: If you leave out of the medium any one amino acid, that amino acid essentially dis- 
appears from the cell pool and falls to levels of less than 0.001 wmoles per ml cells. 
