754 R. W. HENDLER 
structure, then perhaps D- or L-valine could dilute but not serine. For very low 
specificity requirements, all three of the amino acids should be able to dilute the 
labeled valine equally well. In Table I, two such experiments are shown. Expt. I, 
Case 1 shows the situation with high-specific activity radioactive valine (83 ug) 
added to the incubation flask. Case 2 shows the result of adding 6 mg of unlabelled 
L-valine. In Case 3, 6 mg of unlabeled D-valine was added and in Case 4, 6 mg of 
unlabeled 1-serine. The change in specific activity of the intracellular valine pool 
was verified by isolating the amino acid and determining its specific activity. With 
this information and a knowledge of the specific activity of the added valine and 
total uptake of radioactivity into the cells, the dilution of the intracellular pool could 
be calculated for the three situations of decreasing specificity as indicated in Columns 
1, 2 and 3. The change in relative size of the valine pool compared to the other 
amino acids was verified by chromatography of the intracellular supernatant fluid 
on Dowex-50, and these data are shown in Column 4, except in the fourth line where 
the increase is shown for serine. Column 5 shows the relative amount of radioactivity 
recovered in the lipid complexes which were chromatographed on silicic acid, and 
the last column shows the recovery of radioactivity in the most non-polar amino 
acid—lipid complex. Expt. 2 is basically the same. The major differences were that 
both labeled L- and pD-valine were used in the initial case. A larger initial amount of 
labeled valine was used (2 mg each of L- and p- and larger amounts of unlabeled 
valine and 1-serine were used for dilution (20 mg of each). Both experiments show 
that the most non-polar material showed effects of dilution by L-valine, but not by 
p-valine or L-serine. The other lipid components also show distinct signs of specificity 
at the higher concentrations. The increase in radioactivity obtained from the lipid 
complexes upon addition of the D-amino acid may be related to the D-amino acid’s 
ability to raise the pool of L-amino acid slightly by competing with L-valine in other 
reactions of low specificity or by virtue of its having a low competitive affinity 
with respect to the lipids. 
Since total radioactivity is experimentally measured, it is important to take into 
account the increased amount of amino acid in the form of the complex as a conse- 
quence of the higher concentration of amino acid inside the cell as a result of dilution. 
For a monomolecular reaction, where the amount of lipid acceptor is not limiting, 
the total radioactivity held in the form of a complex will be constant even after dilu- 
tion with unlabeled amino acid. If the reaction is multimolecular with respect to the 
amino acid, the total lipid-bound radioactivity will start to increase upon addition of 
unlabeled carrier until the number of multisite lipid acceptors becomes limiting. The 
total radioactivity held will then level off and decrease. If a given lipid fraction can 
accommodate amino acid by two independent reactions, one multimolecular and the 
other monomolecular, and if the number of multimolecular sites become saturated 
early, then the total radioactivity held in the form of a lipid complex will first rise (as 
unlabeled amino acid is added), reach a maximum, start to decrease and level off at 
a value determined by the affinity of the monomolecular site for the amino acid. 
Actually the saturation curves for valine in the lipid complexes in the hen oviduct 
resemble this behavior. The initial phase for the chloroform-eluted material from 
silicic acid behaves like a mixed mono- and multimolecular reaction. The multi- 
molecular site becomes limiting when the external valine concentration is approx. 
7 mM (5 mg added) and the internal free valine pool is approx. 2 mM after a 15-min 
References p. 758 
