--
COMMUNICATIONS 1 U --*1Hb
3550
T,
n----D UllUK
3
vo1.
nn
(23
dioxide, P-methylcrotonate and pyruvate. It was found that P-methylcrotonate and pyruvate were incorporated a t a considerably lower rate than acetate, and that sucrose and carbon dioxide were not incorporated. The dependency of acetate incorporation into rubber upon latex concentration in the incubation mixture is illustrated by the data of Table I. Latex was added to all tubes a t the end of the incubation period and immediately inactivated by the addition of acetone so that equal amounts of rubber were present in all tubes for isolation and counting.
These results suggested that n-a,p-diglycerides might be common intermediates in the biosynthesis of phospholipides and of neutral fat. It has now been found that the same chicken liver enzyme preparations which carry out the synthesis of lecithin also catalyze the enzymatic net synthesis of triglyceride when supplemented with D-a,@-diglyceride and palmitoyl-S-Co A labeled with palmitic a ~ i d - 1 - C ’(Table ~ I). Extensive incorporation of labeled palmitic acid into the triglyceride fraction was noted with the complete system. No significant incorporation was observed if the enzyme was previously boiled. Omission of D- a,&diglyceride TABLE I or substitution of equivalent amounts of palmitate EFFECTOF EXZYME (LATEX)CONCESTRATION OF ACETATE CoA for the palmitoyl-S-Co A reduced incorISCORPORATION ISTO RUBBER‘ poration to low levels.
+
Latex, ml.
Radioactivity (counts/minute/mg. ‘carbon)
0 0.02 .04
TABLE I E X Z Y M A T I C S E T SYNTHESISO F
2.6
2.8 4.5
.06 .08
Experimental conditions
14
38 .10 120 .13 210 a Reaction conditions as for Fig. 1 except for latex concentration a s indicated; incubation time, 4 hours.
1 Complete system 2 Complete system, boiled enzyme
TRIGLYCERIDE Radioactivity Total of tritriglyceride glyceride, fraction, micrototal counts moles
210,000 610 12,200
2.46 .72
D-a-j%Diglyeeride omitted .79 Palmitate Co-1 in place of palmitoyl-S-Co A 3,350 .iil T h e complete system contained 1000 micromoles o f Trib That the radiocarbon to acetate was in fact in- buffer of pH 7.4, 60 micromoles of MgCI?, 40 micronioles of corporated into rubber was demonstrated by deg- D-a,P-diglyceride derived from purified egg lecithin by radation of a sample of the enzymatically syn- treatment with lecithinase of CZ. perf ring en^,^ 1 micromoles thesized rubber to levulinic acid. The specific of palmitoyl-S-Co A labeled with palmitate-l-Cl1 ( 140,001) cts./micromole), 20 mg. of “Tween-20” (polyoxyethylcne activities of rubber bromide isolated as in the above sorbitan m ~ n o l a u r a t e )80 , ~ micromoles of cysteine, and 1.0 experiments, of rubber bromide isolated after pre- ml. of a suspension of mitochondria from chicken Iil-er in liminary alcohol purification of the rubber, and of final volume of 10 ml. T h e tubes were incubated for t\vo prepared hours at 40’. T h e lipides, after extraction from t h e enzyme levulinic acid Zj4-dinitrophenylhydrazone system with ethanol, were taken u p in carbon tetrachloride from the purified rubber were 220, 200, and 230 and repeatedly washed with aqueous ammoniacal ethancil counts per minute per milligram of rubber carbon. to remove unreacted palmitate and palrnitoyl-S-Coh. T h e triglyceride fraction was isolated from the lipid mixture b y FIELDCROPSRESEARCH BRASCH,AGRICULTURAL The radioRESEARCH SERVICE,U. S.DEPARTMENT OF AGRICULTURE t h e chromatographic method of B o r g ~ t r o m . ~ BELTSVILLE, MARYLAND HOWARD J . TEAS activity was measured b y direct plating and counting of :i small aliquot, The total triglycerides were estimated b y DEPARTMEST OF BOTANY ASD PLANT PATHOLOGY t h e hydroxamic method.* MICHIGASSTATE UNIVERSITY EASTLAXSIXG,MICHIGAN ROBERTS.BAXDCRSKI Chemical measurement of the total amount of RECEIVED MAY21, 1956 triglyceride a t the end of the experiment showed T H E ENZYMATIC SYNTHESIS OF TRIGLYCERIDES
Sirs: Previous reports from this Laboratory have described the function of cytidine coenzymes in the biosynthesis of lecithin and of phosphatidylethof lecithin in isoa n ~ l a m i n e . ~The ~ ~ synthesis ~~,~ lated enzyme systems proceeds by the following reactions : CTP
+ phosphorylcholine ++
+
Cytidine diphosphate choline inorganic (1 ) pyrophosphate Cytidine diphosphate choline D-a,P-diglyceride f--f Lecithin 4- C M P (2)
+
Enzyme preparations from chicken liver catalyze the net synthesis of lecithin when supplemented with cytidine diphosphate choline and D-a,P-diglyceride.4 It also has been shown that D-@ diglycerides may be formed by the enzymatic dephosphorylation of phosphatidic acids.4 J O U R N A L , 7 7 , 2.70 (19R-j). 1 2 ) Ici) t < , P Kcwnedy a n d S H W e i J . Bioi Chrm., in prr+ €’ Kennedy, ‘Vu/:I , siilimitteit
(1) E. P . Kennedy :ind S I3 Weiss, l ’ f I I s
3 4
+
that the incorporation of radioactivity was not due to simple exchanges processes, since a considerable net increase of triglyceride was observed in the complete system, compared to the control with boiled enzyme. These results support the conclusion that thc synthesis of triglycerides in these preparations proceeds by the reaction : D-a,B-diglyceride
+ palinitoyi-S-Co Triglyceride f
__f
CoA-SII
(3)
-1 close interconnection is thus indicated between the enzymatic synthesis of phospholipides and of neutral fat.g THEBEN M A YLABORATORY FOR CANCERRESEARCH ASD THE SAMUEL B. IVEISS DEPARTXENT OF BIOCHEMISTRY UNIVERSITYOF CHICAGO CHICAGO, ILLISOIS RECEIVED J u s u 11, 1930 J . Hanahan and R . Vercamer, T w s J U C K X A I . , 7 6 , 1801 ( I ! i s 4 ) ( 8 ) A. Kornberg and W. E. Pricer, Jr., J . B i d . Chenz., 204, 3%!)( l ! G j ) ( 7 ) B . Borgstrhm, Acta Physiol. S c a n d . , 3 0 , 231 (1954). ( 8 ) M . Rapport a n d S . .4lonzo, J . B i d . C h p ~ n, 217, 103 ( I ! l > . j ) . [ ! I ) ’The follctwing :thbrrvi;itiijn.; :ire u i c d in thi, p:iiwr. L~’1.r’ c>-tidinr-.X t r i ~ ~ l i ~ t + l ~ l u iCh1I’ te, = r y t i i l t n t .i~ ’> l i < > \ l ~ l ~ :L ~~
