746 B. AXELROD, J. L. HAINING, T. FUKUI 
as compared to DL-/3-M@C|phenylalanine, 2270 counts/min. In all cases 1.15 wC of the 
amino acids were employed, and their respective amounts were: 0.24, 0.22, 0.36, 
0.39, 0.28 and 0.27 umole. 
There is little doubt about the enzymatic nature of the reactions. The quantity of 
the amino acid—lipid compound formed depends on the quantity of enzyme solution 
used. No product is formed in the absence of enzyme. The activity of the enzyme 
preparation is lost on heating. 
The possibility that the observed reaction might be due to a reversal of the hydro- 
lytic action of a glyceride esterase was tested by adding a 1ogo-fold molar excess of 
glycerol in an effort to depress the formation of the radioactive phenylalanine—lipid 
I MSIEIS, MOE 
INCORPORATION OF [cC]PHENYLALANINE USING 
PURE LIPIDS 
Each vessel contained 1 ml of the soluble enzyme, 5 mg 
of lipid, and o0.4mmole of ptL-[3-!4C]phenylalanine 
(550 000 counts/min) in a final volume of 1.6 ml. 
Incubation was carried out for 2 h. 

(4C) Phenylalanine 

Lipid incorporation 
(Counts/min) 
None 90 
Crude lipid extract 950 
a-Monoolein 16070 
a-Monopalmitin 535 
Triolein 25 
Tripalmitin 25 
Oleic acid 2050 
Palmitic acid 2315 
Lecithin 1725 
* By courtesy of the J. Biol. Chem. 
by the soluble enzyme in the presence of palmitic acid, but the reaction was not 
greatly affected. 
Adding a 50-fold molar excess of unlabeled L-phenylalanine produced virtually 
no depression in incorporation. In order to examine the possible relationship between 
this enzyme and previously described esterases and carboxypeptidase, several specific 
inhibitors were tested. The formation of N-palmitoylphenylalanine from palmitic 
acid and phenylalanine was inhibited 68% by 6.8 - 10-? M_ diisopropylfluorophos- 
phate and 48% by 1.0: 10-3M physostigmine, but not by 2.0 x 10-3 M hydrocinnamic 
acid. The latter is a specific inhibitor of carboxypeptidase. The enzyme solution 
was incubated with the inhibitors for 40 min before the addition of the substrates. 
Crystalline carboxypeptidase was entirely without effect in catalyzing the com- 
bination of pL-[3-C|phenylalanine and palmitic acid, ruling out the possibility that 
an enzyme of this type was responsible for the reaction. 
The product formed from p1-phenylalanine and monoolein was highly purified by 
column chromatography and isolated in weighable amounts. Its specific radio- 
activity on a weight basis agreed with a 1 : 1 molar ratio of phenylalanine to oleic 
References p. 749 
