612 DYNAMIC ASPECTS — PERMEABILITY AND TRANSPORT 
DISCUSSION 
Chairman: HALVOR CHRISTENSEN 
CHRISTENSEN: It should be noted that Dr. HOLDEN’s findings and those with Ehrlich cells are in 
agreement; in neither case is the initial rate accelerated by pyridoxal. At one time we published 
a curve purporting to show initial stimulation of q-aminoisobutyric acid uptake, but we do not get 
this effect with other amino acids nor is it very convincing evidence. 
I understand from Dr. SNELL that pyridoxal added to protoplasts of Stveptococcus faecalis causes 
stimulation of amino acid uptake. Perhaps we can hear from Dr. Mora about that. 
Mora: During studies of the role of the cell wall in the transport of amino acids, we have found 
that pyridoxal stimulates the rate of uptake of glycine only in protoplasts and not in intact cells of 
Streptococcus faecalis. Pyridoxal was used in 1 x 10-3 M concentration and a 30 per cent increase 
in the rate of uptake was observed. 
CHRISTENSEN: The work of Dr. GERHARDT has been mentioned, I wonder if I could ask him to 
comment? 
GERHARDT: A study of bacterial permeability to a metabolically inert amino acid recently has 
been completed in my laboratory by Dr. R. Marguts. lappreciate the opportunity afforded me by our 
convenor to present a generalized summation of this work. Several of its basic premises and results 
may be pertinent to some of the papers presented at this conference. 
First, we sought to meet the need for dissociating uptake of a compound from its subsequent 
metabolism, which enables one to determine just what transport alone involves. This concept has 
been a notable contribution resulting from the studies of Dr. CoHEN AND Dr. Monop, and later 
others, on the “permease” system in bacteria. Analogs of galactosides provided suitable experi- 
mental material, unfortunately not equally met in studies of amino acid permeases. A model of 
amino acid uptake meeting these requirements, however, had been established by the studies of 
Dr. CHRISTENSEN and his associates on the accumulation of methyl analogs in ascites carcinoma 
cells. Following their lead, we investigated q-aminoisobutyric acid (AIB) as a substrate which 
test bacteria might accumulate but not metabolize. 
A second premise of our studies was to provide for direct comparison between bacterial and 
mammalian cells. Employment of a common substrate and a relatable experimental approach in 
two different laboratories we hoped would allow reliable interpretations of similarities and differ- 
ences in the two types of cells. In all too many instances, such comparisons become almost im- 
possible. 
Third, our studies sought to interpret the complex of uptake reactions occurring simultaneously 
in a single bacterium and for a single substrate in terms of known structural features. Bacteria 
have a cell wall in addition to the usual plasma membrane. A basic aim was to identify the perme- 
ability associated with each of these structures rather than the collective surface. In turn, the 
accumulation of amino acid into an internal pool could be distinguished from that into cell wall, 
the latter sometimes operationally termed the “expandable pool”. 
Although Staphylococcus auveus was employed in our intial experiments, a more suitable test 
system was provided by use of Bacillus megaterium strain KM. Using the so-called space technique 
to measure the uptake of “C-labeled AIB, it was found that: (1) AIB is accumulated to significant 
extents by both these organisms; (2) in contrast to most other analogs of natural amino acids, 
ATB is not toxic; and (3) once accumulated, the AIB appears to be inert to further metabolism, 
as shown by chromatographic and other evidence. 
The bacillus accumulated AIB by constitutive mechanisms to a considerably greater extent 
than the coccus and so came to be used exclusively. Under anaerobic conditions, uptake occurred 
by characteristically passive processes of readily-reversible absorption and less-reversible chemad- 
sorption, the cell wall im situ or isolated having a particular affinity. 
Under aerobic conditions, AIB was in addition accumulated by a metabolism-dependent 
mechanism capable of building up an intracellular AIB concentration in excess of one hundred 
times the extracellular concentration. This active transport was stimulated by increasing aeration, 
inhibited by cyanide or fluoride, and uniquely driven from endogenous energy reserves, glucose 
exerting little effect. Active AIB uptake was temperature-dependent (21° optimum, 1.8 Qj) but 
