Ig2, Editor: E. HEINZ 
strongly inhibits protein synthesis in EF. coli. It apparently directly affects the pool forming 
mechanism apart from its interference with synthesis. 
ANDERSON: I would like to ask what the requirements are for your binding sites, because it 
seems to me that if the binding is rather loose no one will isolate this material in a bound form. 
If these sites are more or less ion exchanging sites then they may already have been demonstrated 
sufficiently in experiments that—as far as I know—haven’t been described in detail, but have been 
inferred here. 
The inside of a cell is very hard to reproduce in a laboratory, for this reason. We have a large 
number of poly-anions and low molecular weight cations, so that, for instance, the concentration 
of chloride in liver cells is very low. We would expect under these conditions that cationic sub- 
stances would be bound to the macromolecules present, and this is probably what occurs. When 
we try to make a homogenate generally we have to put in a little bit of salt. But we don’t usually 
make our homogenate under conditions such that the cation is small, and the anion that we add is 
an extremely large one. We would have to add such substances if we wanted to maintain the 
original condition which exists inside the cell. 
So far as the polyamines are concerned, I would expect putrescine, cadaverine, and spermine 
could be bound tightly to RNA and loosely to almost any proteins that are present. Again it 
would be very difficult to decide whether or not there are any specific binding sites for these. 
Cowte: I would like to comment on Dr. ANDERSON’s remarks. Actually we have tried to find some 
binding sites in the cell to account for our data and were forced to some rather unusual conclusions. 
There is so much material in the internal pool of non-exchangeable amino acids that the only single 
component in the cell to complex with this material is the protein. We would like to include the 
binding sites of nucleic acid for this particular pool. Taking the DNA and the RNA of the cell 
alone you would have 3.3 times too many amino acids in the pool per nucleoside residue, so this 
material level obviously cannot be the source of sites. If you add the proteins there are plenty of 
available sites for the pool material. 
BritTEN: Not until this moment, did I realize that we had experiments demonstrating a very 
large amount of binding in cell preparations. This particular experiment deals with a system which 
has a negative heat capacity. It demonstrates that there is a fantastic interaction between water 
and protoplasm. We know, however, very little beyond the crude experimental observations. The 
freezing point of a very thick suspension of EF. coli cells suspended in water was measured. The 
thick goop contained approximately 60° wet cells by volume, and the cells did not need to be 
intact for the phenomenon to occur. An ordinary freezing point system was used with an effective 
motor-driven stirrer, and the temperature was continuously recorded. When the cell was filled 
with water a typical cooling curve was observed. The temperature fell well below 0° and remained 
there while ice steadily froze out. When the cell was filled with the suspension, supercooling was 
observed. However, when an ice crystal was added the temperature suddenly rose to 0° and then 
slowly rose perhaps to as high as + 0.7°. 
HoLpEN: Have you performed freezing point determinations using cells loaded with large 
quantities of proline? 
BrittEN: No. We haven't explored this system, which is obviously a trap of some sort. We have 
done freezing point determinations on released pools in an attempt to estimate the total osmotic 
constituents, and in the case of FE. colz it looks as if, without extensive measurements, the osmotic 
strength within the cell is comparable to that of the medium, and depends on it. This is quite 
distinct from what one would observe in the Gram positive cell. 
HENDLER: Would you go just far enough to explain the relation of the negative heat coefficient 
to the problem? 
BritTEN: Well, the existence of the negative heat capacity shows an interaction between water 
and the protoplasm, because the protoplasm strangely elevates the freezing point of water. It is 
presumably some gel phenomenon that would be very hard to understand. The change in freezing 
point is, for our present argument, the only element of significance, and I hadn’t realized until this 
moment how clearly it demonstrates a gross association of a small molecule with protoplasm in a 
cell preparation. 
ANDERSON: I want to mention some work of Dr. Mazur on freezing of yeast in our laboratory. 
An ice crystal in order to freeze through the pore of a membrane must deform. The freezing point 
of the ice that seeds through therefore, is a measure of the size of the pore. It is a very neat system, 
and yeast cells in distilled water show a survival curve something like this (at blackboard). At 
—5° all the water outside the yeast cells is frozen, and the survival rate is very high. When you 
get down to — 30”, the survival curve drops very suddenly which means that seeding through the 
pores had begun. The functions of the radius of curvature are not ours, but they seem to be pretty 
well worked out. 
ABRAMS: I would like to mention some experiments which indicate that K*+ and Na* ions have 
profound effects on the permeability of bacterial protoplast membranes. Earlier in the discussion I 
presented evidence that the penetration of oligosaccharides and certain amino acids is dependent 
References p. 777 
