ROUND TABLE DISCUSSION 77 
y-aminobutyric acid in the 16,000 x g sediment. Results of experiments by SANO AND Roperts!4 
suggest that sodium ion may be required to bind optimally exogenously supplied y-aminobutyric 
acid. In these latter studies it was shown that isotopically labeled y-aminobutyric acid was bound 
to brain tissue but not to several other tissues which were tested. I think that all available data 
show that the binding of y-aminobutyric acid is fairly specific, but the degree of binding in any 
particular brain homogenate depends on many factors including the molarity, temperature, ionic 
composition and concentration of the suspending medium. This dependence upon the suspending 
medium has made it difficult to determine which cellular fraction has the greatest capacity to bind 
y-aminobutyric acid. In the Ringer solution which SANO AND Roperts used originally, the ™C- 
labeled y-aminobutyric acid was found in the slow speed sediment. On the other hand, in their 
more recent studies in which fractionation of brain homogenate was performed by differential 
centrifugation in a sucrose medium, it was found that most of the isotope was bound by the mito- 
chondrial fraction and less by the slow speed sediment. In these experiments the precipitates were 
resuspended in Tris buffer containing sodium chloride and the binding capacity for “C-labeled 
y-aminobutyric acid was tested by equilibrium dialysis. All of these observations tend to emphasize 
the need for more experimental data with regard to the variables which influence the binding of 
this amino acid to brain tissues. 
GuRorF: Some investigators in our own laboratory have been interested in amines which under 
almost any fractionation conditions are extensively bound to subcellular fractions. But attempts 
to isolate these sedimentable fractions have led to the conclusion that this binding is very non- 
specific. We have done the same type of experiment with tyrosine, using sucrose fractionation of 
spleen. Parts of the free tyrosine did come down in each fraction, but we attached no particular 
significance to this. 
Cowte: I like Dr. ANDERSON’s remarks about the choice of organism and variability of conditions 
which accounts in part for experimental difference. I would like to point out, however, that in the 
yeast, we have at least three states of so-called free amino acids. Referring to Dr. HErNz’s description, 
a certain amount of amino acids may enter the cell through diffusion. In addition material may be 
accumulated and concentrated in excess of the external concentration. In the yeast we have still a 
third form of amino acid, the internal pool which is always present and does not exchange with external 
material and shows functional differences from the other forms of endogenous amino acids. 
Biosynthetic conversions take place in this third pool of amino acids. This pool is insensitive to 
osmotic shock. Thus there are three states of pool amino acids. One of these states, presumably 
that of the internal pool amino acids certainly must represent some form of binding inside the 
cell. 
Ho pen: In addition to the possibility that differences in results may derive from the use of 
different organisms, there is a strong likelihood that different results also may be obtained in a 
given organism depending on whether growing or non-growing cells are studied. You will recall 
that in our experiments with L. avabinosus we found a marked difference in the size of the gluta- 
mate pool and its response to extracellular osmotic strength when cells from early and late ex- 
ponential phase cultures were compared. The proportion of actively dividing cells in these two 
populations would be expected to differ greatly. Amino acid pooling by early exponential phase 
cells had many characteristics in common with the process in growing. E. coli whereas the reverse 
was true of late exponential stationary phase cell. In this connection, I would like to ask Drs. 
BRITTEN and Cowl if they have used non-growing cultures in any of their experiments. 
Cowte: In yeast these different amino acid systems have always been defined during exponential 
growth. Here the formation of the internal pool will be directly proportional to the quantity of 
growth. However just before the cells go into the resting phase the internal pool anticipates this 
condition and you can actually see a different rate of formation of the internal pool before the 
optical density changes. Since, however, I have carefully avoided this condition I can’t say any- 
thing more than this. 
BRITTEN: Our work in general has been with the growing cell, because we have found the in- 
corporation into protein a very useful clue as to what is going on. We have also done experiments 
in the absence of energy sources and at zero degrees, and these do certainly change the pool 
situation. 
In our system, the exhaustion of glucose leads to a behavior of the amino acid pool which, to use 
a far fetched analogy, suggests the presence of a rachet. While glucose is present, external amino 
acids enter the expandable pool. At the moment glucose is exhausted the amount of amino acid 
in the pool stops at the level it has reached and remains stable at this level for long periods. The 
amino acid is not lost from the cells even when you remove the external amino acid in the absence 
(or presence) of glucose. Nevertheless a very rapid exchange occurs in the presence of external 
amino acid. The rate of exchange depends on the amount of amino acid in the pool much more 
than it does on the external amino acid concentration. A generally similar behavior is observed 
when pool formation is suppressed by placing the cells at 0°. 
Beyond these studies we have explored one other example of non-growing cells. Chloramphenicol 
References p. 777 
