614 Chairman: H. CHRISTENSEN 
cause it is quite inert. It is not active in a number of important ways; it is not concentrated, it 
does not act as an inducer, it does not inhibit induction, and it does not inhibit concentration. 
Further, it is not hydrolyzed either by whole cells or by extracts. Therefore, its formation can be 
considered only to be in the nature of a side reaction. 
This, therefore, suggests that the enzyme responsible for the acetylation reaction may be doing 
something else which takes a more direct part in the permeation reaction. In order to try to get 
evidence on this point, we have more recently purified the enzyme and looked at certain of its 
properties. On a biochemical level, these properties do not lend support to the idea that this 
enzyme is a part of the permease reaction mechanism. 
I would like to mention three observations. The first is that the pattern of specificity for the 
acetylation reaction, while similar to that for the accumulation or permease reaction, is in some 
respects vastly different. For instance, phenyl S- and O-glucoside are acetylated, but neither of 
these substrates can be accumulated in the permease reaction. 
Secondly, the apparent Michaelis constant for the permease reaction has been measured for a 
variety of substrates and has been found to be generally in the neighborhood of 10“ or 10° moles 
per liter. In the case of isopropyl thiogalactoside, the Michaelis constant for this substrate in the 
acetylation reaction is extremely high, 3 x 10-1 moles per liter. 
Thirdly, one would expect that this enzyme, if it is involved in the permease reaction, might 
be associated with or be found in the membrane fraction. When protoplasts of E. coli are prepared 
and then shocked with water, the particles have little or no enzymatic activity. The enzyme is 
found in the supernatant solution. Therefore, while evidence based on a correlation in a variety 
of mutants strongly suggests an identity of this enzyme with a component of the permease reaction, 
biochemical evidence so far does not support this view. 
CHRISTENSEN: An important principle in transport studies has been carefully recognized here. 
If a solute is modified to a new form during transport, is this event an essential part of transport, 
or is it merely an incidental event? In informal conversation elsewhere I have heard the present 
work misinterpreted to suggest that an essential transport intermediate has been identified. Dr. 
ZABIN has dealt with this distinction very carefully. 
HENDLER: We have been concerned with the possible participation of the lipid—amino acids in 
transport and in collaboration with Dr. BriTTEN and Dr. R. Roperts at the Carnegie Institute we 
ere in the process now of determining whether we can observe such a role in FE. coli. We are able to 
observe the formation of lipid-soluble forms of amino acid in this organism and to isolate them 
from the cell wall and ribosomes. These complexes turn over rapidly with respect to the amino 
acid. One experiment in particular was rather suggestive. When cells loaded with cold proline 
were exposed to a small amount of highly radioactive extracellular proline, the specific activity 
of the internal pool rose while, the specific and total activity of the external pool was falling. The 
fact that the radioactivity of the lipid-amino acids also decreased during this period indicates that 
they are closer to the external pool in their formation as opposed to being formed from the internal 
pool. 
Dr. DuGGaAN also has been working on this problem in my laboratory, and we have recently 
obtained the mutant mentioned by Dr. HoLpEN. This mutant isolated by Dr. LuBrn at Harvard is 
not able to concentrate proline. The experiments are quite preliminary with this system, but using 
high specific activity, 14C-proline, we are studying the metabolism of the lipid-bound forms of the 
amino acid in an attempt to pick up significant differences in the mutant and wild type. 
Hatvorson: I want to confirm one point made by Dr. Britten; that is, when we have looked at 
the soluble RNA fraction in yeast, we found that it picks up radioactive amino acids at only about 
a fourth or fifth of the rate that we would expect to explain the rate of their incorporation into 
proteins. 
I would like to address a question to Dr. BritTEN. I am intrigued by the fact that he has a by- 
pass for uracil. Together with Dr. CoHEN we have been concerned with a phenomenon in yeast 
where, under some conditions, a similar by-pass appears to exist. I wonder if he would care to 
comment on by-passes for amino acids. 
BritTEN: The evidence for by-pass on kinetic data essentially alone is very clear. Even though 
you pretreat cells with unlabeled uracil, at the moment you add labeled uracil there is incorpora- 
tion into RNA with a delay of less than five seconds. The initial rate corresponds to about 40 per 
cent of the ultimate rate after the main storage pool has come to equilibrium. 
I would like to make two brief philosophical comments. The first has to do with the role of a 
model such as the one that I described. The type of reactions that are postulated which lead to a 
certain set of equations, will not ultimately be lost sight of. They, in fact, in some modified form, 
will enter into the ultimate explanation of proline pool formation. However, the relationship 
between the equations and the object with which they have been associated is quite open. We 
have been unable to find suitable models in which sites are not the principal mechanism for holding 
the pool. But I do not think that we have demonstrated, after considerable effort, that in fact the 
sites are necessary. There are alternate models which may ultimately prove suitable. 
