3316
Vol. 72
COMMUNICATIONS TO THE EDITOR
0.7
t \o
I
I I
\
x
a 150-g. male rat in divided doses twice daily over a period of five days. The rat was kept on the ordinary stock diet. The serine contained a total radioactivity of 2.8 microcuries. The rat was sacrificed and the choline in the liver isolated as the reineckate, then purified through the chloroplatinate, degraded to trimethylamine, which was precipitated as the chloroplatinate and recrystallized, all according to the method of du Vigneaud, et al.' Radioactivity was found in the methyl groups from the choline, SPECIFICACTIVITY PER MILLIMOLE IN COUNTS PER MIX.
0.21
I
I
I
I
I
1
1
I 1
10
30 50 70 Sec. Fig. 1.-Ferrous-ferric exchange: x, radioactivity of ferrous fraction; x - , equilibrium value; Ti/,= 44 * 4
-Platinum, %Calcd. Found
nL-Serine Choline chloroplatinate Trimethylamine chloroplatinate
2 . 2 x 107 2 . 3 X lo4
31.7
31.5
1 . 3 X lo4
37.0
37.0
Confirmation of these findings was obtained with another animal. mary of the data obtained is given in Table I. That all of the choline radioactivity is not in All runs were made a t room temperature 23 * 2'. the trimethylamine fraction indicates that serine, The results indicate that catalysis by traces of a t least in part, has been converted to ethanolchloride was not an important factor in deter- amine; this is additional support for the findings mining the rate; possible effects of other un- of Stetten.4 detected impurities are, of course, not eliminated. There is reason to believe that pteroylglutamic The change of half-time with iron concentration acid and possibly vitamin BIZ may be involved shows the reaction to be second order, presumably in these transformations. first order in ferric and in ferrous iron, in which The finding of radioactivity in the choline case the rate constant in 0.4 f perchloric acid is methyl group demonstrates the in vivo synthesis 16 mole-l-l.-sec.-l a t 23'. of labile methyl groups from the 0-carbon of Experiments on the various factors which affect serine and hence5from the a-carbon of glycine. the rate are being continued. The authors wish to thank Dr. Louis DeSpain Smith for his valuable advice. CHEMISTRY DEPARTMENT BROOKHAVEN NATIONAL LABORATORY After this work had been completed we became RICHARD W. DODSON aware of a report by Sakami6 and Welch and UPTON,N. Y. RECEIVED MAY19, 1950 Sakami' on the incorporation of the methyl group of acetone and of formate into methionine and choline methyl groups. We have obtained similar THE I N VIVO SYNTHESIS OF LABILE METHYL results with radioactive formaldehyde. GROUPS sec.
Sir :
( 4 ) Stetten, J . B i d Chcm., 144, 501 (1942). (5) Sakami, i b i d . , 118, 519 (1949).
I n the course of an investigation into the (6) Sakami, Federation Proc., 9, 222 (1950), abstract. metabolism of amino acids, evidence has been (7) Welch and Sakami, i b i d . , 9, 245 (1950), abstract. obtained which can best be explained on the BIOCHEMICAL RESEARCH FOUNDATION SICURDUR JONSSON assumption of an in Vivo synthesis of labile methyl DELAWARE WILLIAM A. MOSHER groups in the rat. These methyl groups may be NEWARK, RECEIVED MAY15, 1950 derived from the a-carbon of glycine, directly, or indirectly through the @carbon atom of serine, or both, and therefore from serine itself. This THE SYNTHESIS OF THE METHYL GROUPS AND is contrary to the belief that labile methyl groups ETHANOLAMINE MOIETY OF CHOLINE FROM SERINE AND GLYCINE I N THE RATL cannot be synthesized in the animal organism but must be provided in the diet.1,2 Sir : Serine labeled in the 0-position with C1* In an investigation of the mechanism of formawas synthesized according to the method of tion of the phospholipid bases it was found that King.* A total of 20.1 mg. of ~~-serine-D-Cl*L-serine is a source not only of the ethanolamine in 10 ml. of water was given intraperitoneally to portion of choline, but also of its methyl carbon (1) d u Vigneaud, Cohn, Chandler, Schenck and Simmonds, J . atoms. When L-serine labeled with N15 in the Biol. Chcm., 140, 625 (1941). amino group and C1* in the 0-carbon atom was (2) d u Vigneaud, THIS JOURNAL, 72, 1049 (1950), did demonstrate the incorporation of methyl groups from methanol into choline; methanol, however, is not a normal dietary constituent. (a) King, i b i d . , 69, 2738 (1947).
(1) This work was supported by a grant from the American Cancer Society, recommended by the Cornruittee on Growth of The National Research Council.
July, 1950
COMMUNICATIONS TO THE EDITOR
3317
fed to rats the choline from the internal organs2 was extensively labeled in the p- and methyl O H in ] carbon atoms [ ( C H I ) ~ + N - C H ~ - @ C H ~and the nitrogen.a From Table I it can be seen that the isotope dilutions for all three positions are approximately the same. A smaller incorporation of CI4 into the a-carbon of choline also took place. It would seem that P-labeled serine can give rise to a,P-labeled serine which is then converted to ethanolamine labeled in both carbon atoms4 Further investigation is continuing t o explain this finding. TABLE I ISOTOPEDISTRIBUTION IN CHOLINEFOLLOWING ADMINISTRATION OF L-SERINE AND GLYCINE Isotope concentration Isotope concentration p r e c u r s o w - -in cholineActivity in Compound N15 labeled Nitro- u-Car- @-Caradministered concn. carbon Methyl gen bon bon atom c. p. c. p. atom c. p. c. p. %ex- m.a X m.O X % ex- m." X m.a X cess 10-8 10-8 cess 10-8 10-3 3214, Nlk-serine' 2 5 . 8 2.3 10 8 1,060 10.1 0.277 2-Cl4, Nl'-glycinec 21 13,000 4.0 .041 17.5 4.2 l-Cl4, Nlr-glycined 2 7 . 6 389 0 .430 0 0 -in
Counts per minute per dish (2.5 sq. cm.) of carbon a t infinite thickness. Fed 0.47 mM. per 100 g. of body weight per day for two days. Fed 0.40 mM. per 100 g. for one day. Fed 1.45 mM. per 100 g. per day for two days.
The a-carbon and nitrogen5 of glycine are also available for the formation of the methyl groups and ethanolamine moiety of choline, while the carboxyl carbon of glycine is lost in this process. It is apparent that the utilization of glycine does not involve the reduction of the carboxyl groupe5 The isotope dilutions per millimole of precursor fed being greater in the glycine feeding, it is reasonable to conclude that glycine is converted to serines prior to its use for the synthesis of ethanolamine. A point of interest is the nearly equal activity in the P-carbon atom and methyl groups of choline following the administration of either glycine or serine.' The ability of the rat to synthesize methyl groups from the P-carbon atom of serine is in agreement with the recent report8that diets devoid of methyl group donors will support growth when folic acid and vitamin Blz are present. DEPARTMENT O F BIOCHEMISTRY ARTHURWEISSBACH COLLEGE OF PHYSICIANS AND SURGEONS COLUMBIA UNIVERSITY DAVIDELWYN@ NEW YORK,h-.U. DAVIDB. SPRINSON RECEIVED MAY15, 1950 (2) Details of the isolation and degradation procedures will be published elsewhere. (3) Stetten, J . Bioi. Chcm.,144, 501 (1942). (4) Elwyn and Sprinson, $bid., 184, 465 (1950). (5) Stetten, ibid., 140, 143 (1941). (6) Sakami, i b i d . , 178, 519 (1949). (7) Following the completion of these experiments it was reported by Dr. H. G.W o o d (Harvey Lecture, N . Y.,February, 1950) that the s9nthesis of labile methyl groups in the rat from the methyl carbon of acetone and from formate has been observed by Dr. W. Sakami. ( 8 ) Bennett, Science, 110, 689 (1949). ('4) Life Xarurracc Msdlc*l Rsaearch Student R o l l e r , 194O-XOM).
' Thymine DNA
' ~
methyl ring' Cytosine Adenine Guanine
5,610 25,000 748 272 17,800 13,800
3,070 13,760 405 697 27,300 26,000
