766 EDITOR: E. HEINZ 
classical observation. The issue is, I think, to decide to what extent within a given cell the amino 
acid is actually free in solution, and to what extent it is bound, and how this binding is connected 
with special metabolic processes. 
I would like to cause a little confusion about the nature of an osmotic membrane. Anyone who 
has used Dowex-50 ion exchange resin, 2% cross-linked, knows that he takes the risk of blowing up 
his chromatograph tubes because of the large volume changes that occur when concentrated 
aqueous solutions of sucrose or glycerol are passed over the resin. The resin has no membrane in 
the biological sense, but a molecular structure which corresponds to the resin itself. I think it 
would be possible to make models with this resin that behaved osmotically like certain classes 
of cells, with the clear knowledge that no membrane in the traditional sense exists. 
The experiments of Dr. ABRAMS and Dr. SIsTROM are quite suggestive that the amino acid is in 
solution if we ignore for the moment the confusing issue of the nature of solution and of the 
osmotic situation just mentioned. However, I think it is difficult to transfer this evidence to the 
undamaged cell. 
When spheroplasts are made from E. coli by treatment with lysozyme followed by a change in 
sucrose concentration from 10%—5%%, they commonly appear in the phase microscope as large 
transparent spheres with a dark saddle of about the volume of the original cell. The relationship 
between the spheroplast and the original cell is uncertain, in my mind, particularly with regard 
to the location of the original cellular fluids. In any case the clear area is a large trapped volume 
with osmotic properties. According to E. BoLTON’s experiments the capability of the spheroplast 
to form amino acid pools is identical to that of the original cell. The rate of pool formation, final 
pool size and rate of incorporation into protein at a moderate “C-proline concentration are un- 
changed. The spheroplasts, however, can not be treated roughly and their pool is sensitive to 
washing with buffer. In any case the time course and the size of the proline pools are identical for 
these two structures in spite of the fantastic change in the organization of the free water inside 
the cell, or internal osmotic solution, or whatever it is. 
Cowie: If one measures in yeast or in £. coli the accumulation of free amino acids as a 
function of external concentration, a complex incorporation curve is obtained. At low external 
concentrations the accumulation may greatly excede the external concentration. At higher exter- 
nal concentrations the incorporation appears to be proportional only to the external level. 

free 
amino 
acids diffusion 
accumulation component 
log external concentration 
Obviously there is an accumulation mechanism which saturates and which is independent of a 
second process (presumably diffusion) dependent directly upon external concentrations. The total 
quantity accumulated is, in my estimation, contained in two states: a free, and a bound form. 
In yeast the quantity of tyrosine or guanine which can be concentrated exceedes the solubility 
of these compounds in the culture medium. One interpretation of such data is that some of the 
accumulated material complexes with internal substances thereby changing their solubility 
characteristics. There would not be free amino acids or bases. On the other hand some of the 
accumulated material certainly would be in the free state. 
CHRISTENSEN: I am not sure that I can at this moment help to explain the form of Dr. CowIE’s 
curves. It seems to me that one can accept an asymptotic approach to a horizontal line, represent- 
ing a state of saturation of a transport process, only when one measures rates, preferably initial 
rates, rather than the steady state distributions. The steady-state distribution represents the 
algebraic sum of all fluxes. One might have a second transport site mediating a transport, perhaps 
References p. 777 
