AMINO ACID POOL FORMATION IN Escherichia coli 607 
mean distance the carrier must diffuse between taking up an amino acid and deliver- 
ing it to a site is much smaller than this. However, the molecular weight is probably 
much larger and the diffusion constant is probably much smaller in the protoplasm 
than in water. Thus the turnover number probably would be considerably less than 
1000 per carrier per second, and therefore the number of carriers greater than 40 per 
cell. If the number of carriers were as small as this, it would certainly be difficult to 
observe the carrier complex directly. 
The properties of “cryptic” mutants have had an important place in discussion of 
bacterial concentrating mechanisms’. They have not, so far, been mentioned since 
we have limited ourselves to amino acid concentrating systems. For our purposes, 
the observations may be summarized as follows: There are strains of E. COM (Vn, ZH, 
i-) which contain large quantities of the enzyme /-galactosidase, but will not utilize 
lactose or concentrate galactosides. The enzyme is fully active in preparations of 
these cells treated with toluene. Undamaged cells will only split the test-substrate 
(ONPG) at low rates when it is supplied at high concentrations. Other strains 
which utilize lactose contain both the enzyme and an operative concentrating 
system. The enzyme in these cells will split ONPG at high rates whether or not 
treated with toluene and when the concentration of galactosides has been blocked 
by metabolic inhibitors. 
The fact that the mechanism for the concentration of galactosides and the enzyme 
6-galactosidase are controlled by distinct genetic loci is consistent with the carrier 
model. Clearly the carrier, the sites and the enzyme would be three distinct elements 
in the cell. However, in the carrier model no osmotic barrier limits the rate of entry 
of substrate. What then limits the rate of splitting of the substrate (ONPG) by the 
enzyme present in the cryptic mutant (y~, z+, 1~)? An additional hypothesis is neces- 
sary: In the organized cell, the free substrate does not have full access to the enzyme, 
but when associated with the carrier, it can reach the active site and be attacked at 
maximal rate. In defense of this hypothesis, it may be pointed out that there are a 
large number of examples of enzymatic reactions which are suppressed or absent in 
whole cells but occur at high rates in disrupted cell preparations. In some cases, the 
suppression has been attributed to an impermeable barrier but there are a number 
of examples for which the substrate is known to be present in the cell and such an 
explanation is clearly invalid. The fact that (in y+ strains) metabolic inhibitors 
block the concentration process but do not reduce the rate of splitting of ONPG 
further implies that the carrier complex is formed rapidly without the participation 
of energy donors. 
Conclusion 
In this paper we have outlined the rather diverse set of experimental observations 
of the amino acid concentrating processes in E. coli. Any satisfactory model of these 
processes must be formulated with all of this information in mind. 
We have reviewed the simple “permease” and simple “stoichiometric site” models 
and pointed out their failures. Then we have developed the “carrier” model in some 
detail and demonstrated that it can be made to correlate almost all of the data in 
a highly satisfying manner. 
While it is tempting to do so, it should not be asserted that the function of sites 
References p. 609 
