580 J. T. HOLDEN 
amino acids. ABRAMS, who initially observed an energy-dependent entry of sucrose 
into S. faecalis protoplasts accompanied by swelling! 2, subsequently found that a 
variety of amino acids used at high concentrations as osmotic stabilizers also cause 
swelling of protoplasts? (c/. p. 615). Since amino acids within the protoplasts cause 
an influx of water measured indirectly as swelling (7.e. change in optical density) it 
is inferred that they must be free and not bound to adsorption sites. In such experi- 
ments the amino acids were present extracellularly at extremely high concentrations 
and, therefore, moved down a concentration gradient into the protoplasts. A com- 
parable phenomenon has been observed in this laboratory using protoplasts accumu- 
lating amino acids in opposition to concentration gradients (Fig. 4). ABRAMS also has 

OPTICAL DENSITY 

(0) 20 40 60 
MINUTES 
Fig. 4. Swelling of S. faecalis protoplasts associated with glutamic acid accumulation. Incubation 
at 37° ino.18 M phosphate buffer (pH 6.5) containing 0.6 MW sucrose. Optical density was measured 
at 640 mu. Suspensions received supplements at the following concentrations: 1, no additions; 
2, L-glutamic acid (0.003 MW); 3, glucose (0.005 WM); 4, glucose and glutamic acid. 
observed this behavior. WACHSMAN AND STORCK"™ have described energy-dependent 
swelling of B. megaterium protoplasts induced by propionate, various amino acids 
and other organic acids. Recently MArguts’® has shown that protoplasts of B. 
megaterium swell while accumulating a-aminoisobutyric acid. These experiments are 
comparable to those carried out earlier by S1strom!®? with E. coli spheroplasts in 
which swelling was induced by accumulation of galactosides, and they suggest 
strongly that the intracellular amino acids are osmotically active. In none of these 
studies, however, has it been conclusively demonstrated that the osmotically active 
material is the accumulated solute and not some other displaced substance. However 
remote this possibility may seem, this concept will not be secure until the osmotically 
active material is conclusively identified. Furthermore, bacterial protoplast swelling 
induced by accumulated amino acids so far has not been studied quantitatively. In 
view of the evidence that amino acid pools in some organisms may be heterogeneous, 
the possibility must be considered that only a portion of the pool participates in the 
swelling phenomenon. Furthermore, explanations can be conceived for the pheno- 
menon other than that the penetrating solute induces an osmotic influx of water. 
For example, there is the possibility that membrane stability and resistance to 
distention is diminished by adsorption of accumulated acidic substances to the inner 
References p. 592/594 
