Leucine was synthesized with C14 in the carboxyl group. 10 mg. of the radioactive amino acid (DL) and 0.66 gm. (wet weight) of guinea pig liver homogenate were added to a reaction mixture containing 1.3 per cent of an amino acid mixture corresponding to the composition of casein and 0.005 M fumarate, all in a final volume of 4 ml. of isotonic saline solution(1) at pH 7.4. The reaction was carried out under oxygen for 6 hours at 38°

Borsook, Henry

Deasy, Clara L.

Haagen-Smit, A. J.

Keighley, Geoffrey

Lowy, Peter H.

English

Caltech Authors - Main

ISOLATION OF A PEPTIDE IN GUINEA PIG LIVER HOMOG- 
ENATE AND ITS TURNOVER OF LEUCINE” 
sirs: 
Leucine was synthesized with Cl4 in the carboxyl group. 10 mg. of the 
radioact,ive amino acid (DL) and 0.66 gm. (wet weight) of guinea pig liver 
homogenate were added to a reaction mixture containing 1.3 per cent of an 
amino acid mixture corresponding to the composition of casein and 0.005 M 
fumarate, all in a final volume of 4 ml. of isotonic saline solution1 at pH 7.4. 
The reaction was carried out under oxygen for 6 hours at 38”. 
After precipitation of the proteins by boiling at pH 5.0, the non-protein 
filtrate was chromatographed on starch with a solvent mixture consisting 
of 1 part 0.1 N H(‘1,2 parts of N-propanol, and 1 part of N-butanol accord- 
ing to the method of Moore and Stein.2 A radioactive fraction was found 
which emerged from the column before the leucine. This fraction was 
concentrated and purified by chromatography on a smaller starch column. 
The distribution of the color on treatment with ninhydrin and the radio- 
activity gave a single zone of coinciding ninhydrin color and radioactivity. 
The fraction was hydrolyzed with boiling 20 per cent hydrochloric acid 
for 24 hours, the hydrolysate chromatographed on starch, the fractions 
analyzed wit,h ninhydrin, and their radioactivity determined as before. 
Whereas before hydrolysis only one ninhydrin zone was obtained, after 
hydrolysis there were many zones. All the radioactivity was in the leucine 
fraction. 
Once the point of emergence of t,he peptide had been determined by its 
radioactivity, the non-protein filtrate of guinea pig liver (prepared without 
prior incubation or any additions) was chromat,ographed. A peptide was 
found which emerged in the same position as did that’ from the incubated 
digest. 
The identity of the amino acids giving the different ninhydrin zones after 
hydrolysis was det,ermined by chromatography on filter paper3 with known 
amino acids as references. The presence of the following was established : 
* This w-ork is part of that done under contract with the Office of ?;aval Research, 
Cnited States Xavy Department. 
The Vi4 used in this investigation was supplied by the Monsanto Chemical Com- 
pany, Clinton Laboratories, and was obtained on allocation from the I‘nibed States 
Atomic Energy Commission. 
1 Borsook, II., and Dubnoff, J. W., J. Biol. Chem., 171, 363 (1947). 
2 Moore, 8., and St,ein, W. II., Fedex&on Proc., 7, 174 (1948). Stein, W. II., and 
Moore, S., Federation Proc., 7, 192 (1948). 
3 Consden, R., Gordon, A. II., and Martin, A. J. I’., Biochem. J., 38, 224 (19443. 
1041 
1042 LETTERS TO THE EDITORS 
alanine, aspartic acid, glutamic acid, glycine, leucine, methionine, phenyl- 
alanine, and proline. Other amino acids are present ; complete amino acid 
analysis awaits collection of a sufficient amount of the peptide. 
The leucine “turnover,” estimated from the radioactivity of the leucine 
obtained aft’er hydrolysis of the peptide, was 40 per cent in 6 hours. 
We have obtained evidence by methods similar to the above of other 
peptides in guinea pig liver. 
The authors are indebted to and wish to thank Dr. S. Moore and Dr. 
W. H. Stein of The Rockefeller Institute for Medical Research for their 
generosity and courtesy in giving the details of their method of starch 
chromatography and ninhydrin calorimetry prior to publication. 
William G. Kerckho$ Laboratories of th,e Biological 
Sciences 
California Institute of Techn,ology 
Pasadena 
HENRY BORSOOK 
CLARA L. DEASY 
A. J. HAAGEN-SMIT 
GEOFFREY KEIGHLEY 
PETER H. LOWY 
Received for publication, May 14, 1948 


Isolation of a peptide in guinea pig liver homogenate and its turnover of leucine

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Isolation of a peptide in guinea pig liver homogenate and its turnover of leucine

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