BIOSYNTHESIS OF PROTEIN 641 
the radioactivity of the TCA-soluble fraction was shown to be contained in threonine. 
The remainder was found in isoleucine, an amino acid that derives four of its carbons 
from the four carbons of aspartic acid. This isoleucine and a small fraction of the 
total pool threonine are believed to be components of the internal pool since the 
threonine and the isoleucine in the proteins of the cell both became equally radio- 
active?. 
3. Unlike the internal pool the material in the expandable pool is sensitive to 
osmotic shock, extractable with cold water, and readily exchanges with exogenous 
amino acids?. 
Diffusion 
In addition to the material accumulated by the concentrating system amino acids 
enter the cell by diffusion. This process is most easily observed when the concen- 
trating system becomes saturated (or is absent) and amino acids (or their analogs) 
contained in the cell increase directly in proportion to an increase in exogenous 
levels’. Indeed, as shown by HALVORSON AND COHEN, exogenous amino acids 
could be used preferentially for protein synthesis without equilibrating with pre- 
formed expandable pool material. 
Thus at least three processes describe the means by which amino acids become 
available for incorporation in the internal pool: (a) endogenous formation of family 
head amino acids; (b) concentrating or permease system; (c) diffusion. 
At high external concentrations of amino acids the probability of an endogenously 
synthesized family head amino acid entering the internal pool is very small. The 
accumulated amino acids in the expandable pool, and those which have diffused 
into the cell, are at such high levels that dilution of the endogenous material is 
great. In fact with both E. coli! and C. utzlis®, the addition of exogenous {!#C]amino 
acids (approx. I mg each amino acid/ml medium) in medium containing randomly 
4C-labeled sugar results in the appearance in the medium of large quantities of 
(4C]glutamic acid, [!4C]jaspartic acid and [!4C]alanine. Significantly only traces of !C- 
labeled end-product amino acids such as threonine, isoleucine, methionine, proline 
and arginine appear in the medium. Apparently the amino acid production system 
continues to synthesize #C parental amino acids despite the large excesses of [12C]- 
amino acids present. 
While a simple mass-law effect on equilibria is satisfactory as an explanation for 
the competitive reactions which occur between exogenous amino acid and fructose 
carbon, other explanations are necessary to account for the fact that accumulated 
amino acids do not rapidly equilibrate with internal pool material. It has been 
suggested that the amino acids contained in the znternal pool form amino acid— 
protein or amino acid—nucleic acid—protein complexes which provide for this dis- 
tinction? 4. The large quantity of pool material precludes any small molecule—amino 
acid association. Only the macromolecules of the cell are present in sufficiently 
large amounts to provide for a single substance with which the bulk of the in- 
ternal pool may be associated. 
Furthermore, metabolic transformations have been observed among the individual 
amino acids of the pool. During incorporation of C from the fructose of the medium 
the “heads” of each “family of amino acids” become radioactive before the other 
family members; later when the source of #C is removed from the medium these 
References p. 645 
