DISCUSSION 615 
The last point I would like to make is that there has been considerable discussion of free amino 
acids, and I do not really believe that this term has any meaning in the present state of knowledge. 
The amino acid present in a bacterial cell, for example, is dissolved in material which is not only 
25 percent protein, RNA, and lipid but is also highly organized. Under these circumstances the 
amino acid is not in solution in the classical sense. 
CHRISTENSEN: I should like to comment on that point. It seems to me that by working at levels 
of the order of 1078, 10~®, or 10-4 molar, one provides the maximum opportunity for binding to 
obscure the potentiality for uphill transport. These low solute levels must have the best chance to 
be of the same order as the levels of the coenzymes, and it seems to me not so strange that one 
would find intracellular amino acids at these levels bound to coenzymes or holoenzymes. Multiple 
pools are most to be expected under these conditions. This may be an unavoidable difficulty for a 
given organism. But when we come to cases where amino acid levels of several hundred milli- 
molar are obtained, or where gradients of that magnitude are obtained, then the opportunities 
are greatly reduced for sufficiently significant binding or changes of activity coefficients to occur. 
Also, in the experiment Dr. OXENDER has done in our laboratory in which cells were arranged 
into the form of a membrane, so that two extracellular phases could be studied, application of 
conditions stimulatory to transport from one side or the other produced concentration gradients 
whose reality are very hard to question. Although normal secretion perhaps already does this, the 
experiment seems to me to show that the production of real concentration gradients has to be 
reckoned with. 
BritTEN: It seems quite obvious that at low concentrations there are indeed opportunities for 
association of a variety of small pools with sites. The question that I was directly raising was with 
regard to very large pools. In this case, it is not easy for me to visualize the type of affinity with 
the protoplasm which might take place. Part of the difficulty stems from the fact that we do not 
know enough about solution chemistry in dense protoplasm. However, it was precisely with regard 
to very high concentrations that I made my comment. 
Asrams: I would like to make a few remarks with regard to downhill penetration of solutes into 
bacterial protoplasts of Streptococcus faecalis which may bear on this question. These protoplasts 
have no endogenous energy metabolism. If they are suspended in high concentrations of compounds 
which do not penetrate the membrane, the protoplasts are stable; they do not swell. On the other 
hand, if protoplasts are suspended in solutes which penetrate, they will swell and eventually lyse. 
The rate of swelling in a particular solute is an indication of the rate of penetration. 
We have examined the swelling or lack of swelling of S. faecalis protoplasts suspended in a great 
variety of amino acids and, as one might expect, there has been a wide variation in the rate of 
penetration. 1-Alanine penetrates, as indicated by the rate of swelling, at a very rapid rate. On the 
other hand, p-alanine penetrates only at a very slow rate. L-Serine penetrates at a rather rapid 
rate, somewhat less than L-alanine, but its optical isomer penetrates only very slowly. L-Alanine 
penetrates far faster than f-alanine; a-aminobutyric acid penetrates much more rapidly than 
y-aminobutyric acid. We have studied the behavior of approximately twenty amino acids in this 
way. All I want to point out is that with regard to spontaneous downhill penetration, the mem- 
brane appears to be able to recognize the difference between closely related amino acids. Further- 
more, with L-alanine and L-serine, we see a lag period in the rate of penetration, which would 
indicate that some preliminary reaction must precede penetration. 
One further remark with regard to the composition of isolated cell membranes of S. faecalis may 
be of interest. This membrane contains a large amount of lipids, as has been found with other 
membranes from bacteria. In addition, it contains a small amount of RNA, and since the proposi- 
tion has been made that RNA might be involved in some manner in the transport process, I sug- 
gest that the presence of RNA in the membrane would be eminently suited for such a role. 
GurorFF: Could you comment, Dr. CHRISTENSEN, on the maintenance of pools in ascites cells with 
respect to absence or presence of energy sources. 
CHRISTENSEN: The Ehrlich cell has very great endogenous reserves of energy, apparently in the 
form of lipid, so that this cell is able to continue to produce energy for a great many hours. The 
cell also has a considerable glycolytic capacity, so one cannot, by inhibiting either respiration or 
glycolysis alone, prevent amino acid accumulation. Apparently, one has to strike at both of these 
things at once. Under anaerobic conditions adding glucose or fructose is necessary if the accumula- 
tion is to continue. Eventually the pH falls enough through the formation of lactic acid to produce 
a handicap. Accumulation continues experimentally as long as we have cared to follow amino acid 
uptake. I think, Dr. Hernz has attempted to exhaust the energy supplies of the cells by various 
means. Would you care to comment on this same question? 
He1Nnz: Unfortunately, we did no experiments to exhaust the energy supply. We did, however, 
try to wash all the glycine out of the cell but without success. A certain amount, somewhat less 
than ro millimoles per liter, always stays inside the cell. Also, the kinetic data indicate that 5 to 10 
per cent of the cellular glycine may not be directly exchangeable. This could represent the “internal 
pool”, but I do not think one can say more about it. 
