102 j- 2 HOLDEN 
Another approach to the demonstration of osmotic activity by intracellular amino 
acids is found in experiments showing that bacterial protoplasts swell during uptake 
of amino acids. ABRAmMs! (also cf. discussions this Symposium) has shown that S. 
faecalis protoplasts swell when they glycolyze in the presence of high concentrations 
of some amino acids used as osmotic stabilizers. Experiments demonstrating the 
swelling of protoplasts during accumulation of carbohydrate derivatives!® and ali- 
phatic acids!8* have been presented as evidence of the intracellular osmotic activity 
of these substances. Comparable observations have been made in this laboratory 
using amino acids®’. All such studies, however, suffer from the defect that the identity 
of the osmotically active molecule is not known. In no case has it been excluded that 
the entering solute does not displace intracellularly some substance which is then 
responsible for the osmotic activity. 
A number of studies have shown that the size of the bacterial free amino acid pool 
can be controlled by extracellular osmotic activity (cf. Table IV; HOLDEN, BRITTEN, 
this Symposium). In gram-positive bacteria extremely large pools normally are 
retained even during water washes at which time concentration gradients between 
intra- and extracellular compartments of several thousand-fold would appear to exist. 
However, in gram-negative bacteria the pool is much more sensitive to loss under 
these conditions. Furthermore, the pool in gram-positive bacteria becomes osmotically 
labile when the amount of cell-wall substance is reduced slightly (8°. 8& HOLDEN, 
p- 582). While such observations demonstrate the osmotic sensitivity of a cell structure 
responsible for retention of the amino acid, they do not identify it as the cytoplasmic 
membrane and, therefore, do not justify a definitive comment on the intracellular 
state of the amino acid. 
There is a strong possibility that microbial amino acid pools are heterogenous. In 
yeast, COWIE AND MCCLURE were able to distinguish a concentrating pool in which 
extracellular amino acids initially accumulate in a form very sensitive to loss by 
exposure to solutions of low osmotic strength, and a smaller internal pool which con- 
tains internally synthesized amino acids as well and which is less sensitive to osmotic 
shock. BRITTEN using E. coli also has suggested the occurrence of two proline pools 
which differ in specificity for this amino acid. The highly specific pool may be related 
to the similarly specific pool described by LAcHs AND Gros to be associated with 
soluble RNA. 
In conclusion, certain knowledge concerning the intracellular state of the free 
amino acid pool in microorganisms has not yet been achieved. It is reasonable to ex- 
pect that some part of it will be entrained in the protein synthetic apparatus. There 
is serious question, however, whether more than a small fraction is bound in this 
way. 
FUNCTION OF THE POOL AND APPLICATION OF POOL STUDIES 
A large number of studies have shown that the cellular protein composition is un- 
related to the composition of the free amino acid pool. However, it is now well estab- 
lished that the amino acid pool in microorganisms provides precursor material for 
the synthesis of protein. This and related aspects of pool studies form a separate part 
of this Symposium and will not be discussed further here. In some organisms the pool 
appears to have other uses as well. Dawes AND Hortmes*® showed that in Sarcina 
lutea the pool provides the direct substrates for maintenance of endogenous respira- 
References p. 105/108 
