In a previous communication it was shown that choline and betaine are effective in promoting methionine synthesis from homocysteine in tissue homogenates (1). Data presented in this paper indicate that dimethylthetin, (CH3)2+SCH2COO-, which has been shown by Welch (2) to be lipotropic and has been reported by du Vigneaud (3) to promote growth on a methionine-free, homocysteine-containing diet, is 20 times as active as betaine in methionine formation. Dimethyl-β-propiothetin, (CH3)2+S(CH2)2COO-, recently isolated from Polysiphonia fastigiata by Challenger and Simpson (4) is also highly active. The enzyme for this transmethylation is found in the liver and kidney of all animals tested. Its high activity and general distribution suggest its biological importance in methionine synthesis

Borsook, Henry

Dubnoff, Jacob W.

English

Caltech Authors - Main

DIMETHYLTHETIK AiXD DIMETHYL-p-PROPIOTHETIN 
IN METHIONINE SYNTHESIS* 
BY JACOB W. DUBNOFF AND HENRY BORSOOK 
(From the Kerckhoff Laboratories of Biology, California Institute 
of Technology, Pasadena) 
(Received for publication, May 5, 1948) 
In a previous communication it was shown that choline and betaine are 
effective in promoting methionine synthesis from homocysteine in tissue 
homogenates (1). Data presented in thispaper indicate that dimethylthetin, 
(CH&+ SCH&OO-, which has been shown by Welch (2) to be lipotropic 
and has been reported by du Vigneaud (3) to promote growth on a methio- 
nine-free, homocysteine-containing diet, is 20 times as active as betaine in 
methionine formation. Dimethyl-/3-propiothetin, (CH&+S(CH&COO-, 
recently isolated from Polysiphonicz fustigiata by Challenger and Simpson 
(4) is also highly active. The enzyme for this transmethylation is found 
in the liver and kidney of all animals tested. Its high activity and general 
distribution suggest its biological importance in methionine synthesis. 
Methods 
Viobin extracts were prepared by stirring 5 gm. of Viobin’ in 100 ml. of 
water for half an hour and filtering. Fresh extracts were prepa.red from 
organs of animals which had been killed by stunning and thoroughly bled. 
The organs were chilled, homogenized with 2 parts of buffer in the homo- 
genizer of Potter and Elvehjem (5), and strained through cheese-c1ot.h. 
The buffer (6) is composed of 0.0128 M sodium phosphate, pH 7.4, 0.123 
M sodium chloride, 0.005 M pot,assium chloride, and 0.003 M magnesium 
sulfate. 
Methylmercaptoacetic acid was prepared by Dr. M. Fling according to 
the method of Larsson (7). 
Dimethylthetin was kindly given us by Dr. A. D. Welch. 
Dimethyl-fl-propiothetin was prepared by the method of Biilmann and 
Jensen (8). 
m,-Homocystine was prepared from DL-methionine by the method of 
* Presented before the meeting of the American Society of Biological Chemists at 
Atlantic City, March 15-19, 1947. Aided by a grant from the United States Public 
Health Service. 
1 Viobin preparations are commercially prepared desiccated and defat,ted raw 
tissues manufactured by the Viobin Corporation, Monticello, Illinois. 
789 
790 DIMETHYLTHETIN IN METHIONINE SYNTHESIS 
Butz and du Vigneaud (9) and reduced to m-homocysteine as described 
by Riegel and du Vigneaud (10). 
Dimethylglycine was prepared by the method of Schubert (11). 
4 ml. of buffer solution containing the enzyme and substrates were in- 
cubated in 20 ml. beakers in an apparatus especially designed for non- 
manometric studies (12). In this apparatus as many as thirty 20 ml. 
beakers employed as reaction vessels are held in a stainless steel container 
fitted with a cover through which any gas mixture may be passed. The 
container is incubated at 38” and shaken at 90 oscillations per minute in a 
small water bath. 
After incubation the solutions were deproteinized by the addition of 
0.5 ml. of 30 per cent trichloroacetic acid and 1 ml. of water to each beaker. 
With guinea pig liver homogenates after the addition of trichloroacetic 
acid, the solutions were brought to a boil in t,he incubation apparatus in 
order to get filtrates which would remain clear in the analytical procedure. 
Filtrates of other organs offered no difficulty. 
Methionine was determined by a modification of the method of Mc- 
Carthy and Sullivan (13). The procedure was as follows : To 2 ml. of the 
trichloroacetic acid filtrate were added 0.2 ml. of 5 N NaOH followed by 0.1 
ml. of 1 per cent freshly made sodium nitroprusside. The solutions were 
incubated at 38” for 5 to 10 minutes and then 1 ml. of an acid mixture 
consisting of 9 volumes of concentrated hydrochloric acid and 1 volume of 
85 per cent phosphoric acid was added. After 10 minutes the solutions 
were read in a Klett-Summerson calorimeter with a green filter. 
If the solutions are cooled in ice before the addition of strong acid, 
homocysteine reduces the color by an amount which increases with in- 
creasing concentration of methionine. When this cooling step is omitted 
as described, the decrease in color due to homocysteine is a small and con- 
stant value over a wide range of methionine concentrations, and no diffi- 
culty is encountered with gas bubbles during the measurement of the color. 
1 mole of methylmercaptoacetic acid formed by the demethylation of 
dimethylthetin gives a color equivalent to 0.6 mole of methionine in this 
determination. Accordingly, all apparent increases of methionine due 
to the addition of dimethylthetin must be divided by 1.6 to compensate 
for the equivalent amount of methylmercaptoacetic acid formed in the 
reaction. Dimethyl-fl-propiothetin is chromogenic, but it can be de- 
stroyed by allowing the solutions to stand overnight after the addition of 
5 N NaOH. The nitroprusside is added the following day and the de- 
termination continued as described. The chromogenic power of methyl- 
mercaptopropionic acid, the demethylated product of dimethyl+propio- 
thetin, is approximately 0.9 that of methionine on a mole basis. 
J. W. DUBNOFF AND H. BORSOOK 791 
Results 
In rat liver choline, betaine, dimethyl+-propiothetin, and dimethyl- 
thetin show significant activity in methionine formation (Table I). Di- 
methylethanolamine, dimethylglycine, and methylmercaptoacetic acid, 
the compounds formed by the removal of one methyl group from choline, 
betaine, and the dimethylthetin, respectively, are inactive. 
If homocysteine is present in excess, it can be directly shown that only 
one methyl group is transferred per mole of dimethylthetin (Fig. 1). The 
reaction with betaine is too slow to reach equilibrium; only about 0.6 mole 
equivalent of methyl is transferred per mole of betaine in 24 hours. 
TABLE I 
Methionine Formation in Rat Liver Homogenate 
1 ml. of 1:4 homogenate. Homocysteine 25 mg. per cent; all other substrates 
12.5 mg. per cent. Total volume 4 ml.; gas phase, nitrogen; time, 3 hours; tempera- 
ture, 38’. Methionine values are the average of three determinations. 
Reaction mixture M&hi-mine found 
Homocysteine. . . . . . 
“ + choline. . . . . . . 
“ + betaine. . . . . . . . . . 
I‘ + dimethyl-&propiothetin.. 
“ + dimethylthetin. . . . 
‘I + methylmercaptoacetic acid. 
“ + dimethylglycine.. . . . . 
‘I + dimethylethanolamine.. . . 
“ + monomethylethanolamine. 
............ 
............ 
............ 
............ 
............ 
. 
. . 
. . 
. . 
. 
. . 
. . 
. . 
mg. per cent 
1.0 f  0.05 
2.6 f  0.05 
4.2 f  0.01 
4.5* f  0.1 
8.4* f  0.05 
1.0 i 0.0 
1.0 f  0.1 
1.1 f  0.05 
1.3 f  0.2 
* Corrected for chromogenic value of demethylated product as indicated in section 
on methods. 
Table II shows the distribution of the betaine- and dimethylthetin- 
transmethylating enzymes and approximate &m&r&nine values for VariOUS 
tissues. Only liver and kidney show activity with betaine and dimethyl- 
thetin. 
The effect of pH on the activity of the dimethylthetin enzyme is shown 
in Fig. 2. The optimum pH is about 7.8. 
Neither the betaine nor the dimethylthetin enzyme is inactivated by 
simple dialysis. 
The formation of methionine from dimethylthetin, dimethyl$-propio- 
thetin, betaine, or choline was not inhibited by cyanide, azide, arsenate, or 
arsenite. 
792 DIMETHYLTHETIN IN METHIONINE SYNTHESIS 
The two enzymes can be distinguished by the greater stability of the 
dimethylthetin enzyme at pH 4.0 as shown in Fig. 3. The variation of the 
0 I 2 3 
TIME HRS. 
FIG. 1. Number of methyl groups transferred per mole of dimethylthetin. 1 ml. 
of 5 per cent solution of Viobin liver in buffer (6); dimethylthetin 5 mg. per cent; 
L-homocysteine 12.5 mg. per cent. Temperature, 38”; gas phase, nitrogen. 
TABLE II 
Distribution and Activity of Melhionine-Forming Enzyme Systems 
The figures are &methionine; average of two determinations; probable error f5 per 
cent. 
Each vessel contained 1 ml. of homogenized guinea pig or rat tissue diluted 1:4, 
or 1 ml. of a 5 per cent Viobin solution. nL-Homocysteine 25 mg. per cent, betaine 
or dimethylthetin 12.5 mg. per cent. Total volume, 4 ml.; gas phase, nitrogen; 
time, 3 hours; temperature, 38”. 
I 
Guinea pig 
I 
Rat I Hog (Viobin) 
-I 
-- I- ~~ 
Liver........... 1.5 0.09 / 17 1.5 0.11 
Kidney. . 0.3 0.06 ’ 5 0.2 0.03 
Spleen. . 
Muscle. _. 0 0 
Pancreas. 
: Dirt$yl- Dimeth- - ylthetin Betain’ 
BeQUIlt? I I 
0 0 
I i 0 0 
ratio of &dimethylthetin to &b&ine (Table 11) also points to two different en- 
zymes for the two methylators. 
The dimethylthetin enzyme may be purified free of betaine enzyme and 
concentrated about 100-fold by precipitating a 5 per cent aqueous solution 
J. W. DUBNOFF AND H. BORSOOK 793 
of Viobinl liver with alcohol. The fraction precipitated at between 11.5 
and 18 per cent alcohol contains almost all the activity of the whole ex- 
tract. While the crude homogenate is effective with homocystine and 
Oi 
6.0 65 7.0 7.5 6.0 6.5 
PH 
FIG. 2. Effect of pH on methionine formation from dimethylthetin. 1 ml. of 5 per 
cent Viobin; dimethylthetin 12.5 mg. per cent, nL-homocysteine 12.5 mg. per cent. 
Temperature 38”; gas phase, nitrogen; time, 1 hour. 
FIG. 3. Stability of enzyme to PH. Upper curve, dimethylthetin transmethylase; 
lower curve, betaine transmethylase. 5 per cent Viobin allowed to stand 24 hours at 
5” at given pH, and relative activity at pH 7.5 determined at 38”. Substrates 12.5 
mg. per cent. Gas phase, nitrogen; total volume, 4 ml.; time, 3 hours. 
homocysteine, this fraction reacts only with homocysteine. Although the 
dimethylthetin transmethylase can be prepared free from the betaine 
transmethylase by further alcohol fractionation, all active betaine trans- 
methylase preparations have had dimethylthetin transmethylase activity. 
794 DIMETHYLTHETIN IN METHIONINE SYNTHESIS 
Whether the latter enzyme is required for betaine activity is still un- 
certain. 
An attempt was made to demonstrate the presence of dimethylthetin in 
animal tissues. Neutral and acidified aqueous and alcoholic extracts of 
Viobinl pancreas, spleen, kidney, and liver, fresh beef pancreas, and guinea 
pig and rat liver and kidney were inactive with a partially purified di- 
methylthetin enzyme. Liver and kidney homogenates of the rat and 
guinea pig were allowed to stand at pH 4.0 at 5” for 18 hours to destroy all 
but the dimethylt,hetin methylating system. Any increase in methionine 
on addition of homocysteine in such a system could be considered as evi- 
dence of preformed dimethylthetin. No increase in methionine occurred. 
These experiments seem to exclude any significant quantity of preformed 
dimethylthetin in these tissues. On the addition of labile methyl donors 
to homogenates there is a slight synthesis of methionine in most experi- 
ments, suggesting the presence of small amounts of preformed homocys- 
teine. 
None of these reactions is reversible under our conditions; i.e., methi- 
onine will not remethylate dimethylglycine, dimethylethanolamine, or 
mercaptoacetic acid aerobically or anaerobically in the presence of high 
energy-yielding metabolites. These reactions were studied by measuring 
the change in methionine concentration in the presence of these putative 
methyl acceptors. 
Although dimethylthetin is very effective with homocysteine, it will not 
methylate glycocyamine in rat or guinea pig liver homogenates. 
DISCUSSION 
Evidence has been presented that there are at least four compounds 
which can furnish methyl groups for methionine synthesis in tissue homo- 
genates. These compounds, dimethylthetin, dimethyl+-propiothetin, 
betaine, choline, are all “onium” compounds characterized by the coordina- 
tion of an additional methyl group to sulfur or nitrogen, and they all react 
in the absence of oxygen or energy donors. It has been directly demon- 
strated in these and previous studies (1) that the methyl groups in di- 
methylglycine, dimethylethanolamine, and methylmercaptoacetic acid are 
not transferred under conditions in which a methyl of the “onium” com- 
pound is labile. This confirms the findings in feeding experiments on the 
availability of methyl in dimethylglycine and dimethylaminoethanol (14). 
The methyl of methionine, which is held by a covalent bond to sulfur, 
but is nevertheless labile, requires energy for its transfer to glycocyamine 
(15) and to nicotinamide (16). 
Du Vigneaud and his collaborators have proved rigorously that the 
methyl groups of choline, betaine, and methionine constitute a dietary 
J. W. DUBNOFF AND H. BORSOOK 795 
“pool” of physiologically interchangeable methyl groups. The evidence 
reported here indicates that in the tissues there does not exist a “pool” 
of labile methyl groups in the sense that the transfer is directly between 
any two members of the dietary pool of labile methyl compounds. It seems 
likely that rather than a “pool” there are specific methyl donors for each 
methyl acceptor (i.e., the methylation of glycocyamine by methionine 
(15)), and that a given methyl compound may be related to another only 
indirectly through a series of methyl transfer reactions.2 
We have so far failed to find any methyl transfer reaction which is 
reversible in the usual chemical sense. Methionine, for example, does not 
directly remethylate dimethylethanolamine, dimethylglycine, or methyl- 
mercaptoacetic acid. 
The present findings indicate that the physiological transfer from me- 
thionine to choline must be a cyclic process in which some and possibly 
all steps are irreversible. In some stages of the cycle oxidation and pre- 
sumably, therefore, energy is required ; other stages may proceed anaero- 
bically. In such a dynamic state a given labile methyl-containing com- 
pound cannot be utilized or depleted to the same extent by all methyl 
acceptors. This is in accord with the fact that not all methyl donors are 
equally effective in overcoming growth inhibition by such compounds as 
glycocyamine (17, 18) and nicotinamide (19, 20). 
The effectiveness of dimethylthetin suggests its importance in the bi- 
ological synthesis of methionine. Its apparent absence from animal tissues 
may be due to its rapid demethylation in the presence of homocysteine, or 
it may be present and function in only catalytic amounts. The occur- 
rence of dimethylthetin in the dietary sources has not been studied. Its 
homologue, dimethyl+propiothetin, has been isolated from algae (4) and 
may be present in pineapple (21). This compound may, therefore, prove 
to be more important biologically than dimethylthetin despite its lower 
activity in methionine synthesis. 
The authors wish to acknowledge the assistance in this work of Miss I. 
Silberbach. 
SUMMARY 
1. An enzyme has been isolated in a partially purified state which trans- 
fers a methyl group from either dimethylthetin or dimethyl-/?-propiothetin 
to homocysteine. 
4 The data presented here do not establish a direct methyl transfer from all four 
compounds to homocysteine. Unpublished evidence suggests that choline is first 
oxidized to betaine. Whether the latter compound transfers directly or through a 
methylthetin derivative has not yet been determined. 
796 DIMETHYLTHETIN IN METHIONINE SYNTHESIS 
2. Dimethylthetin is 10 to 20 times as effective as betaine as a methyl 
donor in tissue homogenates. Dimethyl-@-propiothetin is more effective 
than betaine. 
3. The enzyme is found in the liver and kidney of rat, guinea pig, and 
hog, but is absent from muscle, pancreas, and spleen. 
4. The reaction proceeds until one methyl group has been transferred 
from dimethylthetin to homocysteine. Mercaptoacetic acid is inactive. 
5. Homocystine is completely inactive as a methyl acceptor in purified 
extracts. 
6. The reaction is independent of O2 and is not inhibited by oxidative 
poisons. 
7. The dimethylthetin transmethylase is distinguished from the betaine 
transmethylase by its stability at pH 4.5. 
8. The possible role of dimethylthetin and dimethyl-p-propiothetin in 
the biological formation of methionine is discussed. 
BIBLIOGRAPHY 
1. Borsook, H., and Dubnoff, J. W., J. Biol. Chem., 169, 247 (1947). 
2. Welch, A. D., private communication to Moyer, A. W., and du Vigneaud, V., J. 
Biol. Chem., 143, 373 (1942). 
3. du Vigneaud, V., Harvey Lectures, 36, 39 (194233). 
4. Challenger, F., and Simpson, M., Piochem. J., 41, p. xl (1947). 
5. Potter, V. R., and Elvehjem, C. A., J. Biol. Chem., 114, 495 (1936). 
6. Cohen, P. P., and Hayano, M., J. Biol. Chem., 166, 239 (1946). 
7. Larsson, E., Ber. them. Ges., 63, 1347 (1930). 
8. Biilmann, E. I., and Jensen, K. A., Bull. Sot. chin., 3, 2306 (1936). 
9. Butz, L. W., and du Vigneaud, V., J. Biol. Chem., 99, 135 (1932-33). 
10. Riegel, B., and du Vigneaud, V., J. BioZ. Chem., 112, 149 (1935-36). 
11. Schubert, M. P., J. BioZ. Chem., 111, 671 (1935). 
12. Dubnoff, J. W., Arch. Biochem., 17, 327 (1948). 
13. McCarthy, T. E., and Sullivan, M. X., J. BioZ. Chem., 141, 871 (1941). 
14. du Vigneaud, V., Chandler, J. P., Simmonds, S., Moyer, A. W., and Cohn, M., 
J. BioZ. Chem., 164, 603 (1946). 
15. Borsook, H., and Dubnoff, J. W., J. BioZ. Chem., 132, 559 (1940); 171, 363 (1947). 
16. Perlzweig, W. A., Bernheim, M. L. C., and Bernheim, F., J. Biol. Chem., 150, 
401 (1943). 
17. du Vigneaud, V., Chandler, J. P., Cohn, M., and Brown, G. B., J. BioZ. Chem., 
134, 787 (1940). 
18. Stetten, D., Jr., and Grail, G. F., J. BioZ. Chem., 1’ , 175 (1942). 
19. Handler, P., and Dann, W. J., J. BioZ. Chem., 146, 357 (1942). 
20. Stekol, J., Abstracts, Biological Division, American Chemical Society, Memphis, 
I39 (1942). 
21. Haagen-Smit, A. J., Kirchner, J. G., Deasy, C. L., and Prater, A. N., J. Am. 
Chem. Xoc., 67, 1651 (1945). 


Dimethylthetin and dimethyl-β-propriothetin in methionine synthesis

Abstracts, Biological Division,

private communication to

http://authors.library.caltech.edu/12251/1/DUBjbc48b.pdf

Dimethylthetin and dimethyl-β-propriothetin in methionine synthesis

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main