2782
DC
T‘IGNEAUD,VERLY,WILSON,RACIIELE,RESSLER AND KINNEY
l C O V r R I R I T I 0 U FROV 1[IF I l E P A R T l t E U T O F
Yol. 7 3
BIOCHEMISTRY, CORNELL UKIVERSITY MEDICAL COLLEGE]
One-Carbon Compounds in the Biosynthesis of the “Biologically Labile” Methyl Group BY
VINCENTDU VIGNEAUD, KALTER G. L.VERLY,’~ JOHN E. WILSON,JKLIAN R. RA4CHELE,CHARLOTTE RESSLER.4ND JOHN h l . K I N N E Y ’ ~
Studies with methanol, formaldehyde and sodium formate labeled with CI4 have shown that these substances can be utilized by the rat in the formation of the “biologically labile” methyl group. This has been demonstrated by the injection of these substances a n d the isolation of the choline from the animal tissues In addition, the carbon of the formyl group of C’4-formyl-~-phenylalanineand of the methyl group of C1%nethyl stearate has been shown to appear in the methyl group of choline. Experiments with methanol doubly labeled with deuterium and C14in the methyl group have indicated that the methyl group of methanol was not being utilized, a t least t o any great extent, directly in transmethylation but was b:ing utilized through oxidation ,itid subsequent reduction to the labile methyl group in the biosynthesis of the latter
In 1945 i t was reported2 from this Laboratory that synthesis of “biologically labile” methyl groups occurred somewhere in the bodies of rats kept on an apparently adequate diet, although, as i t was pointed out in the communication, the data did not distinguish between direct synthesis by the tissues and synthesis by intestinal bacteria with subsequent utilization of the methyl groups in the tissues. Indications of the synthesis of this grouping had been obtained earlierIaatbwhen it was noted that some rats would grow with no known source of labile methyl group. While the crucial experiment in collaboration with Professor Reyniers, Dr. Luckey and their co-workers a t LOBUSD of the University of Kotre Dame was under way to determine whether or not this synthesis of labile methyl groups could occur in germ-free rats,“ we undertook a program of studying the possible precursors of the methyl group, for whether the synthesis took place in the tissues of the rat or through bacterial synthesis in the intestinal tract, the data would be of biological significance. As a result of the germ-free rat experiment4 and of the in vitro work of Sakami and Welch5-8 (vide infra), we now know that the labile methyl group can be synthesized by the tissues of the rat, and consequently our results in this paper on the onecarbon precursors of the labile methyl group are undoubtedly attributable in most part to synthesis of this grouping in the tissues. The single carbon compounds labeled with C14 which we have investigated included methanol,ga bicarbonateIgb and formate.“b The bicarbonate was not found to act as a precursor of labile methyl groups. In this connection, i t might ( I ) (a) Fellow of the Belgian American Educational Foundation. !b) A. IS. C. Post-doctoral Fellow of the Sational Research Council. ( 2 ) d u Vigneaud, Siiniimnd\ Chandler and Cnhn .I 8101.C ’ h t i i ! , 119, 753 (1945). ( 3 ) (a) du Vigneaud. Chandler, hloyer and Keppel. J . H i d L‘hr 131, 57 (19301; ( b ) Toennie., Bennett and IIecle.;. Cvrmth. 7, 231 (1943). (4; This work demonstrated t h a t the synthesis o f the methyl group took place in the germ-free rat and hence was not dependent upon the presence of intestinal bacteria. .2 preliminary announcement of these results with a general discussion of the biological synthesis of methyl groups has been presented (du Vigneaud, Ressler and Rachele, . S L ~ P ? ? T P , 112, 267 (1950)). ( 5 ) Harvey Lecture by €I G. Wood, February 16, 19.50. (6) Sakami, Fcderntioiz Proc., 9, 222 (19.50), J . B i d Chem., 187, 369 (1950). ( 7 ) Welch and Sakami, Federation Proc., 9, 245 (1950). In t h e oral presentation of this paper, they reported t h a t formaldehyde was a precursor of labile methyl groups in cilro. (8) Sakami and XVelch, J . B i d . Chevi., 187, 379 (1950). (9) Preliminary reports of these results have already appeared: (a) d u Vigneaud and Verly, THIS J O U R N A L , 72, 104!1 (1950); ( I ) ) A n Vigneaud, Verly and IV’ilson, ibid , 72, 2819 (1950).
be pointed out that Siekevitz and Greenberglo have shown that carbon dioxide is not reduced to formate by rat liver slices. I n the case of methanol we found that the carbon was incorporated into the methyl groups of choline isolated from the tissues of the rat after the subcutaneous administration of C1*-labeled methanolga The earlier observation of Binkley and that methyl phosphate appears to be utilized in the methylation of guanidoacetic acid by rat liver homogenates is also of interest in this connection. We next turned to experiments with formaldehyde and formate and found that the carbon of these compounds likewise appeared in the methyl group of the choline isolated.gb The experimental results are summarized in Tables I, I1 and 111. While this work was under way, studies by Sakami* with Ci”-methyl-labeled acetone showed that the C14 appeared in the methyl groups of choline and methionine, probably via the formation of “formate.”’2 Furthermore, Welch and Sakami’J demonstrated that the synthesis of the labile methyl group occurred in rat liver slices and in the whole rat from formate. Since the results of Sakami,6 of Welch and Sakami,’ and of our laboratoryya)bwere reported, there have appeared confimiatory data regarding methanol and formate by Arnstein, l 3 formaldehyde by Jonsson and llosher,l” and formate by Siekevitz and Greenberg.’% Both i ~ o t o p i c l ~ -and ~ ’ growth on the synthesis of the methyl group from metabolic precursors of “formate” such as glycine and serine likewise are in harmony with the utilization of “formate” in the synthesis of the methyl group. Evidence from the work of Handler, Bernheim and Klein and from this Laboratory has shown that formalc]ehyde‘21 --?R and formic are intermediates 1101 Siekevitz and Greenberg, J . Biol. Chenz., 180, 84.5 (1949). I 1 1 ) Ainkley and Watson, h i d , 180, 071 (1949j. ( 1 2 ) Sakami and Welch have used “formate” t o indicate either formate itwlf or a one-carhon intermediate of formate metabolism ( 1 3 ) Amitein, Biocht.i!i J , 4’7, s v i i i (19.50). ( 1 4 ) Jonsnon and hlosher, THISJ O U R N A L , 72, 3316 (1950). (13) Siekrritz and Greenberg. J . Bzol. Cham., 186, 275 (1950). i l ( j ) IVeissbach, E l n y n and Sprinson, THISJOURSAL, 72,3316 (1950) (17) Elwyn and Sprinson. i b L . , 72, 3.717 (1950).
( I S ) Stekol, Bennett, Weiss. Halpern and Weiss, paper presented before the American Society of Biological Chemists, Atlantic City, 2;. J . , .4pril 17, 1950. (19) Stekol, Bennett, Weiss, Halpern and Joralemon, Abstracts of Papers, 118th Meeting of t h e American Chemical Society, Chicago, September 3 , 1950. ( 2 0 ) Stekol and Weiss, J . Biol. C h e w , 186, 343 (1950). ( 2 1 ) Handler, Bernheim and Klein, i $ i d . , 138, 211 (1941). (22) Mackenzic, Kacheie, Cross, Chandler and du Vigneaud, i b i d . 183, 617 (1950). ( 2 3 ) Ilackenzie, i b i d . , 186, 351 (1950).
June, 1951
BIOSYNTHESIS OF “BIOLOGICALLY LABILE”METHYLGROUPS
2783
in the oxidation of the methyl group of sarcosine. results demonstrate that the methyl radical in Since Horner and MackenzieZ4have demonstrated methanol is not used in the same way as the methyl that the methyl groups of methionine and betaine group of methionine in transmethylation and inare sources of the carbon of the methyl group of dicate the strong possibility that the methyl sarcosine, the intermediates in the oxidation of the radical of methanol is being utilized, a t least in methyl group of sarcosine become of significance large part, as a precursor of a biologically labile to the general question of the oxidation of labile methyl group via “formate” during which process methyl groups.2zj26 Furthermore, the results ob- deuterium is partially lost. I n the experiments already discussed the comtained by SakamiZ6 indicated that the methyl group of choline contributes to the formation pounds were injected. We thought that it might of “formate” in the rat, and Siekevitz and Green- be of interest to supply a source of labile methyl berg15 have provided evidence that the methyl groups via the intestinal tract which would present groups of methionine and choline are used in the to the body a continuous supply over a period of formation of formate in vitro. Thus, with form- time in contrast to the injection of the labile aldehyde and formic acid as intermediates in the methyl precursor which furnishes the source of oxidation of the labile methyl group, the biological labile methyl group over a shorter period. It was conversion of these oxidation products to the decided to use a methyl ester for this purpose and labile methyl group takes on particular significance. Cl4-methyl stearate was employed. The degree The appearance of the carbon of methanol in the of incorporation of C1* into the methyl group of choline of the animal body allows several interpreta- choline in this instance, however, was approxitions. It might be serving as a donor of intact mately the same as in the case of the injected methyl groups for the synthesis of one of the com- methanol, formaldehyde or formate. In order to determine to what extent the formyl pounds in the transmethylation series. It is furthermore conceivable that methanol might be an group of a formyl amino acid might act as a preintermediate in certain transmethylation reactions, cursor of the labile methyl group, C’*-formylthat is, an actual metabolic intermediate in the phenylalanine was injected. There was no protransfer of the methyl group in toto. On the other nounced difference between the results with this hand, the methanol might be acting as a precursor compound and those already described for formate. of the methyl group through oxidation and subExperimental sequent reduction, and thus might arrive in the Labeled Compounds.-The C14-labeledbarium carbonate, transmethylation series viu this indirect route. C14-labeled methanol, and the mixture of C14-labeled methWe felt that this question might be approached anol and formaldehyde were obtained from the Oak Ridge by studying methanol doubly labeled in the methyl National Laboratory, on allocation from the United States group with deuterium and C1*. It has already been Atomic Energy Commission. Sodium C14-Biearbonate.-Barium C14-carbonate was demonstratedz7 that in transmethylation from in water and decomposed with phosphoric acid. methionine doubly labeled in this way, the methyl suspended The liberated C14-carbon dioxide was trapped in aqueous group can be transferred to choline without appreci- sodium hydroxide. After an aliquot had been removed for able change in the ratio of deuterium to C14j2* radioactivity determination, the remaining solution was If, in the case of doubly labeled methanol, the titrated with hydrochloric acid to a faint pink with phenoldeuterium to C1*ratio in the methyl group of the phthalein. C14-Methanol.--Sodium bisulfite was added to a mixture choline isolated from the rat were the same as that of radioactive formaldehyde and methanol in aqueous in the methyl group of the injected methanol, the solution and the methanol was distilled in vacuo. After a methanol must have played a role only in actual second distillation the distillate was tested with dimedon and found t o be aldehyde-free. transmethylation. If, however, the ratio of deu- (dimethyldihydroresorcinol) C14-Formaldehyde.-Ordinary methanol and formaldeterium to C1* in the methyl group of the choline hyde were added as carriers t o a mixture of radioactive methwere less than that in the methyl group of the anol and formaldehyde in aqueous solution. Sodium bisulinjected methanol, then a t least some of the meth- fite was then added and the solution was distilled to dryness in vacuo. The residual formaldehyde bisulfite compound anol must have arrived in the choline via oxidation and excess bisulfite were washed with methanol and then and reduction to a newly formed methyl group. dissolved in water. After addition of about one-half volume The deuterium to C14 ratio in the methyl group of of methanol the solution was distilled a t reduced pressure. Formaldehyde was regenerated from the residue by adding the choline isolated was considerably lower than hydrochloric acid and passing oxygen-free nitrogen through that in the methyl group of the methanol injected, the solution for several hours t o flush out the sulfur dioxide. as shown in Table IV. It is apparent that these The solution was neutralized with sodium hydroxide. (24) Horner and Mackenzie, J . Biol. Chem., 187, 15 (1950). (25) Mackenzie, Chandler, Keller, Rachele, Cross, Melville and du Vigneaud, ibid., 169, 787 (1947); Mackenzie, Chandler, Keller, Rachele, Cross and du Vigneaud, ibid., 180, 99 (1949). (26) Sekami, ibid., 179, 495 (1949). (27) Keller, Rachele and du Vigneaud, ibid., 177, 733 (1949). (28) In this earlier experiment the ratio of deuterium to C14 in the methyl group of the choline was calculated from the isotopic content of the isolated choline on the assumption that the deuterium (du Vigneaud, Cohn, Chandler, Schenck and Simmonds, J. Biol. Chem., 140, 625 (1941)) and C’4 were located in the methyl group. In a comparable experiment recently completed, the choline was isolated and degraded to trimethylamine. The ratio of deuterium to C14 in the trimethylamine w a s the same, within experimental error, as that in the methyl group of the methionine fed. Thus the methyl group had been transferred i n 1010.
Sodium C14-Formate.-This material was prepared from the barium C14-carbonate by conversion to sodium bicarbonate and reduction of the bicarbonate by hydrogen with palladium as a catalyst by a method previously described ~ ,was ~ ~ difor the reduction of potassium b i c a r b ~ n a t e . ~It luted with a suitable amount of carrier and used directly. C14-Deuteriomethanol.-An aqueous solution of methanol labeled in the methyl group with deuterium and C14 was obtained by mixture of ordinary water, deuteriomethanol and CJ4-methanol. The C14content of the mixture, as given in Table IV, was determined by combustion, precipitation of the carbon dioxide as barium carbonate and determination of the radioactivity. The deuterium content of the methanol was obtained by application of the falling drop method (29) Melville, Rachele and Keller, J . B i d . Chem., 169, 419 (1947). (30)’Bredig and Carter, Ber., 47. 541 (1914).
nu VIGNEAUD, VERLY,WILSON,RACHELE, KESSLERAND KINNEV
"TS4
V O ~i .3
TABLE I
ADMINISTRATIONOF LABILEMETIIYL PRECURSORS Rat
No.
712 894 723 759 968 969 816 804 808 851
8
we., g..
181 213 174 208 210 223 180 194
Diet
I
Compound injected
SdHC"On SaHCLsOO:; C "H::OH C'4DaOI) C"DsO1) C'AD301) Ci4H20
Molarity of injected solution
Number of subcutaneous injections
0.025 ,032 .7.5 2.58 3 .G 3.45 0.93 093 ,095 ,095
1 15 9 4 15 15
Millimoles injected
Duration6 of expt., days
0.13 3 0.49 5 8 I 6.75 5 8 I 9.8 2 8 11" 34,
