COMPOSITION OF MICROBIAL AMINO ACID POOLS IOI 
cerned with amino acid transport and pool turnover. Since these subjects are discussed 
thoroughly elsewhere in this Symposium, only brief mention will be made here of the 
conclusions reached in such investigations. 
Conditions required to liberate the pool uniformly are consistent with the hypo- 
thesis that it is retained by a peripheral or intracellular membrane, although none of 
the studies taken alone would exclude the possibility of retention within an intra- 
cellular particle or attachment to intracellular polymers. Bacterial pools can be 
liberated by many relatively mild procedures including: (a) shaking with glass beads 
in the cold; (b) freezing and thawing; (c) grinding with abrasives; (d) sonication; 
(e) osmotic lysis of protoplasts; (f) treatment with detergents, cold trichloracetic or 
perchloric acid, boiling water, warm ethanol or cold butanol; (g) extremes in pH 
such as 1.5 or 10.5. It is obvious that in addition to destroying hypothetical perme- 
ability barriers such treatments expose intracellular structures to foreign and poten- 
tially disruptive osmotic and chemical environments, so that the possibility cannot be 
excluded confidently, that the pool is retained in association with intracellular poly- 
mers. It is of interest that a number of investigators have studied cells washed with 
acetone®*: 166 and lyophilyzed cells subsequently incubated in buffers*®? and found 
them to retain an amino acid pool. 
CHESBRO AND Evans*? have found that the release of amino acids from Strepto- 
coccus faecium is dependent on hydroxyl-ion concentration and that the kinetics of 
amino acid appearance in the extracellular buffer and disappearance from the pool did 
not coincide. It is apparent that more than one process was involved since, in addition, 
much larger amounts of glutamic and aspartic acids disappeared from the pool at pH 
10.5 than appeared in the extracellular phase. Furthermore, a number of amino acids 
such as glycine, serine, threonine, ornithine and y-aminobutyric acid appeared in the 
extracellular pool even though their levels in the intracellular pool did not appear 
to change. Of course, such experiments are not comparable to the procedures normally 
used to liberate pools where metabolic change is avoided as much as possible, but there 
is a possibility that they might help to establish the mechanism of pool retention. 
CowlE AND McCiure* have shown that exposure of yeast to high hydrostatic 
pressures increases the ease with which the pool contents can be removed. Since such 
leakage is dependent on a supply of exogenous carbohydrate, it appears that exit 
of the intracellular amino acids is not simply a diffusion process through a disrupted 
membrane. However, a direct demonstration of association between pool amino acids 
and intracellular polymers or particles has not been achieved, although LACHS AND 
Gros!6 have reported that a small part (possibly 5°%) of the E. coli pool is combined 
with a soluble nucleic acid fraction. 
In bacteria, MircHELL’® has attempted to demonstrate that the intracellular 
solutes express an osmotic activity which one would expect from their predicted 
concentration in the cell assuming them to be in free solution. This was done by 
equilibrating cell pastes with the atmospheres above sucrose solutions of various 
concentrations and measuring the weight of water taken up by the cells. However, 
the expected contribution to this activity of the amino acid pool would not exceed 
20%, of the total, and the possible error in the method arising from the long incubation 
required is too great to decide with certainty from these data whether or not the amino 
acids are free or bound. A cogent discussion of the evidence bearing on the intracellular 
state of low molecular-weight solutes in bacteria has been presented by M1TcHELL™?. 
References p. 105/108 
