LIPO—AMINO ACID COMPOUNDS 745 
one is always faced with the possibility that a radioactive amino acid can serve as a 
metabolic precursor of a radioactive lipid. 
The lipid which was obtained in incubation of the rat liver whole supernatant 
with [44C|\phenylalanine was separated by silicic acid column chromatography* into 
the phosphatidic and non-phosphatide fractions. The radioactivity appeared in the 
latter fraction. When this fraction was subsequently refractionated the radioactive 
material was eluted after the sterols, triglycerides and sterol esters. 
Chromatography of the radioactive fraction on paper gave but one radioactive 
area and this was quite distinct in position from that expected for phenylalanine. 
Control experiments with phenylalanine showed that there was no tendency for the 
free amino acid to change its mobility in the presence of the lipid material. Hydrolysis 
of the radioactive lipid (6 N HCl, 110°, 18h) released a water-soluble substance 
which was radioactive and migrated coincidentally with free phenylalanine. Milder 
conditions (2 N HCl, roo’, 2 h) liberated only negligible amounts of phenylalanine. 
The radioactive lipoidal fraction also released other ninhydrin-positive substances, 
of varying mobilities presumably amino acids which were not radioactive. Although 
these experiments were carried out with pi-phenylalanine similar results were ob- 
tained with the natural isomer. 
FORMATION OF LIPO-AMINO ACID COMPOUNDS BY “PURIFIED” RAT-LIVER ENZYME, 
SPECIFICITY OF THE REACTION AND NATURE OF THE PRODUCT 
In order to learn more of the nature of the amino-acid incorporating system as well 
as of the product it was necessary to separate the enzyme system from the endogenous 
lipid acceptor. To this end®, rat liver was extracted in a Waring Blendor with acetone 
at —20° and the resulting powder extracted with diethyl ether. Despite the removal 
of considerable lipoidal substance suspensions of the extracted material were still 
effective in catalyzing the incorporation of added [14C|phenylalanine into lipid- 
soluble material without the necessity of additional lipid. Furthermore the enzymatic 
activity remained substantially particle-bound. In order to remove the residual lipid, 
a suspension of the extracted material was allowed to autolyze for 3h at 37° and 
then extracted again with cold acetone. The residue thus obtained exhibited little 
activity until some of the extracted lipid was returned. Also, an appreciable portion 
of the activity could now be extracted with buffer. The activity could be concen- 
trated by ammonium sulfate precipitation; dialysis yielded a product which was not 
only free of endogenous lipid, but was also low in endogenous free amino acids. 
The lipid obtained in the second acetone extraction was separated into phospho- 
lipid and non-phospholipid fractions. Both were effective as “acceptors” when tested 
with phenylalanine. Results obtained with some specific lipids appear in Table III. 
Monoolein, as the a-isomer, was the most effective of the glycerides tested. Triolein 
and tripalmitin were almost without effect. Rather unexpectedly free oleic and 
palmitic acids functioned better than any of the glycerides. Lecithin showed good 
activity. 
In addition to phenylalanine, a number of other amino acids were tested using 
monoolein as the lipid substrate, under comparable conditions. [2-“C}Glycine, 
DL-[1-M@C]leucine, p1-[1-!4C}lysine, pi-[1-!C]valine and pr-[1-4Cjalanine were in- 
corporated in the amounts of 2240, 1685, 825, 770 and 425 counts/min respectively, 
References p. 749 
