764 Editor: E. HEINZ 
is almost identical in some respects with the problem of a chemical reaction. You have somewhat 
similar types of exponential terms involving activation energy. 
As to the problem of fvee versus bound, no solute is wholly free in anything except in the state 
of a dilute gas. It will have, in general, an activity coefficient other than unity, which is determined 
by these energy relations. It will also be hydrated to an extent determined by the activity of water. 
The activity of water depends on all the other components that are around. 
The cases that have been talked about here are extreme cases: On the one hand, the binding of 
the solute you are interested in to some structure, by which you will mean a relatively fixed 
relationship in which the solute and the binding agent come within, less than one Angstrém of 
each other and stay that way; and the free state, which I think is wholly fictitious in solution 
chemistry but which we could represent by considering the solute as being in pure water with 
nothing else around. 
There is evidence that no solute in the liquid system is ever completely bound, and osmotic 
effects will depend on the activity of water and not merely on the presence of discrete membrane 

NO GLUTAMATE 

ABSORBANCY 
0.4 
25 50 75 100 
MINUTES 
Fig. 1. The osmotic effect of metabolic glutamate uptake in protoplasts. Protoplasts of S. faecalis 
were stabilized in solutions of sucrose (0.4 7) —K phosphate (0.075 M, pH 7.2), with and without 
glutamate (0.001 IM). Glucose (0.01 7) was added at zero time and swelling was observed photo- 
metrically. The enhanced swelling in the presence of glutamate may be ascribed to the osmotic 
effect of glutamate uptake. 
phases. It is, therefore, useless to discuss this problem as if we were dealing with an osmometer. 
In most of the cases it is physically and cytologically true that you are not dealing with an osmo- 
meter in the classical sense; that is, a bag with a solution inside and another solution outside, in 
which all effects are due to holes through which some molecules physically can not pass. 
Whatever the relation of molecules to the interface of the cell is, there is no question of there 
being holes that are too small for something to squeeze through. All the effects, in other words, 
have to result from physicochemical forces acting between molecules. Hence there is a gradation 
of possibilities from the idealized completely free to the chemically completely bound. Besides the 
question of comparative biochemistry it would therefore be important to try to answer the question 
of free versus bound: (a) in terms of how much free and how much bound, rather than just yes or 
no, and (b) in terms of a specific mechanism. In other words, we have to ask what in a given case 
may affect the activity of water in such a way as to produce certain osmotic effects in some cases 
but not in others, and whether this is chemically a reasonable hypothesis. 
ABRAMS: I would like to describe some experiments with Streptococcus faecalis protoplasts 
which have a bearing on the present discussion. These protoplasts have no endogenous energy 
metabolism and such metabolism, which is entirely glycolytic, can be introduced by adding glucose. 
The resting protoplasts (no glucose) are osmotically stable in sucrose (0.4 M), thus sucrose is 
essentially impermeable under these conditions. However, on addition of glucose, a swelling takes 
place (Fig. 1) which can be shown to be correlated with the penetration of sucrose as indicated 
by labeling experiments with sucrose-“C. The reversal of swelling takes place subsequent to the 
completion of glycolysis (ABRAms?). If a small amount of glutamate (o.oo1 M) is added to this 
system, an additional swelling takes place, as can be seen in Fig. 1, and it can be shown that during 
this process glutamate is taken up. The additional swelling may be ascribed to the osmotic effect 
of free glutamate concentrated in the protoplasts during glycolysis (ABRAMS AND MACKENZIE’). 
References p. 777 
