770 Editor: E. HEINZ 
In this particular case, the first interpretation that I would give to the fact that the efflux is 
proportional to the total glycine concentration is that if a carrier did mediate the outflow, it 
would not have been saturated in this circumstance. So that in fact the concentration, the number 
of carriers filled with amino acid as amino acid complexes, would be simply larger in one case than 
in the other. 
LoFTFIELD: Then shouldn’t they be proportional to the amount of the bound material? 
REINER: Broadly speaking that would be true, and in this case they are proportional, as I judge 
the statement, to the actual amount of concentrate. Is that correct? This is to say that under the 
circumstances the carrier is not saturated, so that it reflects the amount that is bound. 
Heinz: With our system we never found any saturation phenomena of the glycine efflux, even 
at intracellular glycine concentrations of more than 60 mM. 
GuRoFF: Some observations that we made a couple of years ago with the isolated rat diaphragm 
may be of interest here. In the intact diaphragm, prepared by the technique of KIPNIs AND CorI, 
you observe an endogenous level of 20 wg of tyrosine per gram of tissue. You can incubate this 
diaphragm under various rather unfavorable conditions without removing significant amounts of 
tyrosine even in the presence of metabolic inhibitors. If you expose a diaphragm to large concentra- 
tions of tyrosine, tyrosine enters the diaphragm. If you resuspend the diaphragm in tyrosine-free 
buffer, most of the tyrosine comes out except for an amount comparable to the original endogenous 
concentration. We further observed that the initial rate of tyrosine entrance into the diaphragm 
was proportional to external concentration and not to the difference between this concentration 
and the endogenous 20 wg per g. Since the entrance of tyrosine into diaphragm cells conforms 
largely to the criteria of simple diffusion and does not depend on metabolism the endogenous 
tyrosine or an equivalent amount does not seem to equilibrate with the entering tyrosine. 
ANDERSON: I wanted to mention some experiments on what is in some respects a model system. 
The isolated rat liver nucleus, as shown by the interference microscope or the phase contrast 
microscope, is very permeable to proteins. If you add to the solution a very small amount of 
calcium chloride or magnesium chloride, ‘‘b/ebs’’ begin to appear on the surface of the nucleus, which 
ultimately rise completely off the nucleus, and it is not difficult to show that these “‘blebs’”’ are 
impermeable to protein. It can also be shown that protein put into the nucleus prior to the addi- 
tion of calcium chloride is now inside these “blebs” and does not come out. 
A small amount of sucrose added to this solution prevents these “‘b/ebs’’ from forming, so that the 
membranes seem to have large pores with some mobile lipid component which can flow together 
over these pores. My point is that the membrane is a function of the experimental conditions, so 
that we may be dealing with different cells in different solutions. In other words, the structure of 
the membrane is not a constant. 
I would like to ask whether there are any experimental data on binding of amino acids to 
isolated cell components? Has anyone done simple experiments such as centrifuging these compo- 
nents down to see whether or not the amino acids fall e.g. with soluble proteins, or with micro- 
somes ? 
HEINz: Several such experiments have been carried out with Ehilich cells by Dr. CHRISTENSEN 
and his collaborators, as far as I remember, and by ourselves, but the amino acids appeared 
always in the supernatant, whether the cells had been broken by pressure release or by grinding in 
the frozen-dried state. Dr. CHRISTENSEN also tried then to equilibrate a suspension of broken cells 
within a cellophane bag with normal extracellular medium containing amino acids. Also here the 
final distribution ratio of the amino acids was only slightly higher than unity so that hardly more 
than a negligeable amount of amino acids could have been bound to cellular components. 
Baxter: We have done some simple experiments with y-aminobutyric acid which seem to indi- 
cate that this amino acid is bound to specific cell constituents in brain. We were initially interested 
in localizing, within the cellular components of brain tissue, those enzyme systems which were 
responsible for the synthesis and degradation of y-aminobutyric acid. As a by-product of this study, 
which involved the differential centrifugation of guinea-pig brain homogenates, we found that a 
surprisingly large portion of the intrinsic y-aminobuty1ic acid of these tissues was contained in the 
slow-speed sediment. We have not degraded this fraction any further. 
ANDERSON: How dilute is this preparation? 
Baxter: The medium we used is identical to that recommended by Basrorp for the separation 
of brain mitochondria. It consists of 0.4 MW sucrose supplemented with some heparine and EDTA. 
The homogenate is made up of one part of brain tissue to six parts of medium (w:v). 
ANDERSON: This would have a bearing on it, and also the solution in which the homogenate 
was made would have quite an effect. 
Baxter: The experimental conditions will determine to a large extent whether y-aminobutyric 
acid is bound or unbound in a brain homogenate. ELLiIotT AND VAN GELDER! were the first to 
demonstrate this. They showed that in a brain homogenate suspended in 0.88 MW sucrose solution, 
only 10% of the total y-aminobutyric acid of the tissue was bound to the 18,000 x g sediment. 
A similar brain homogenate suspended in a Ringer-type salt solution retained 60-70% of the total 
References p. 777 
