DISCUSSION 565 
possibility to Dr. GuroFF’s attention, to question whether, in the absence of oxygen, he is limiting 
migration to diffusion. It may well be that mediation is still occurring but without a supply of 
energy to permit a concentrative mediation. 
H. RosENnBERG: Mr. Chairman, I would like to ask you about the involvement of certain che- 
lating mechanisms in the transport of amino acids, which you mentioned briefly. I was wondering 
whether chelators like EDTA have any pronounced effect on the process. One could, for instance, 
imagine a sexadentate chelate of cobalt, with two free positions which could involve a double 
attachment for an amino acid, and provide the correct built-in discriminator for an optical isomer. 
CHRISTENSEN: Through the years we have very seriously considered this possibility. There is 
further evidence in that direction in the fact that the a-amino acids are concentrated, and there 
are mechanisms also for the /-amino acids, but at least for the tumor cells and the other cells we 
have studied, y-aminobutyric and similar amino acids with the amino and carboxylate groups 
further removed, do not fit into these mechanisms at all. Similarly, with the diamino acids, the 
space between the two amino groups is critical so that 5- or 6-membered rings might be formed 
with a metallic ion, for example. Therefore, we have applied two kinds of tests. First we have 
determined the effect of EDTA and other binding agents on transport. These do tend to be 
inhibitory, but the inhibition does not appear at once. Their use is marked by a loss of potassium 
from the cells, and this kind of loss we have generally found to be associated with a gradual decline 
in the ability to transport amino acids, on what basis we are not sure at present. Assuming that 
they have access to the metal, chelators like EDTA and 8-hydroxyquinoline ought to begin much 
more quickly to inhibit uptake if the metal is really serving in the concentration mechanism. 
The other approach has been to take the isotopic forms of these metal ions, put them into the 
environment of the cells in trace amounts, increasing as insignificantly as possible the concentra- 
tion prevailing there, and then seeing if their flux is increased by a heavy load of amino acids 
presented for accumulation. One metal, manganese, has its influx into the Ehrlich cell increased by 
the uptake of a, y-diaminobutyric acid, and in the presence of pyridoxal, by glycine and certain 
other amino acids. This is permissive kind of evidence, but it has no great force in indicating that 
manganese is operating as a carrier, and I have my doubts. The metals cobalt, zinc, and ferric iron 
were instead held out of the cell, as one might expect, by the presence of amino acid. Since the 
amino acid level was perhaps ten times as high inside as outside, obviously the metal was getting 
only to the extracellular compartment and failed to follow the amino acid into the cell, either 
passively or as part of the carrier. 
One never is too sure about the form taken by metals in this part of the periodic table in solutions 
of pH 7.4, so far as ionic activity is concerned. The conditions are rather uncertain and no doubt 
natural chelators escaping from the cell define the final activity of the metal. True, the manganese 
was sometimes as low as 1ro-" molar, but one still cannot be sure if this value represents a real 
activity. Hence, this experiment does not provide a final answer and it is only permissive. 
