ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 185, No. 2, January 30, pp. 443-449, 1978
Oxidation KATHERINE
New
Jersey
Medical
of One-Carbon Compounds to Formate Dioxide in Rat Liver Mitochondria’ F. LEWIS,
School,
College Received
VERRELL WILHELM of Medicine
May
M. RANDOLPH, R. FRISELL’ and Dentistry
27, 1977; revised
of New
September
and Carbon
EDITH
Jerse.y,
NEMETH,
Newark,
New
Jersey
AND
07103
19, 1977
In rat liver mitochondria, swollen with phosphate and supplemented with NAD+, the oxidation of the methyl carbon of sarcosine to formate is enhanced by the addition of NADP+. No carbon dioxide is formed. Formaldehyde and serine, which are the only oxidation products of the methyl group in the absence of the pyridine nucleotides, are decreased by an amount equal to the formate produced. Carbon dioxide, as well as formate, is produced when the mitochondria are treated with EDTA, even without the addition of the pyridine nucleotides. When the mitochondria are exposed to pyrophosphate without added NAD+ and/or NADP+, all of the oxidized sarcosine-methyl can be recovered as formate, [3-Clserine, and carbon dioxide. Formaldehyde accumulates only if the system is supplemented with Mg’+. In the presence of NADP+ or the combined pyridine nucleotides, serine accumulation is depressed by an amount equal to the increase in carbon dioxide production. Both carbons of glycine and the 3-C of serine can also be oxidized to carbon dioxide in the pyrophosphate-treated mitochondria. The oxidation of the methyl carbon of S-adenosylmethionine to formaldehyde, [3-Clserine, formate, and carbon dioxide requires a whole homogenate supplemented with glycine. Neither exogenous formaldehyde nor formate is oxidized to carbon dioxide in any of the mitochondrial systems capable of converting sarcosine-methyl to carbon dioxide. Under conditions in which [NS,N”‘-‘4C-methyleneIand [1L”“-‘4C-formy11tetrahydrofolate can be isolated as intermediate products of [‘4CHnlsarcosine, exogenous lm,A”“-‘4Cmelh.y1erLeltetrahydrofolate can also be converted to [3-“Clserine, [“Clformate, and [‘Xlcarbon dioxide.
In mammalian systems, N-methylglytine (sarcosine) is produced both via the “one-carbon cycle” (2) and by transmethylation between S-adenosylmethionine and glycine (3-5). The N-methyl group is very labile to oxidation (6) and becomes an important precursor for “one-carbon transfer” reactions (7). The three dehydrogenation reactions which produce formaldehyde, formate, and carbon dioxide from the methyl carbon occur exclusively in mitochondria (2, 8, 9). Under certain conditions, the oxidation of the methyl carbon 1 Supported preliminary (1). s Present School of Greenville,
may be restricted to the level of “active formaldehyde” so that the only products are serine and/or formaldehyde (2). However, the implication of folate derivatives in these reactions has not been established unequivocally (10, 11). Subsequent studies have demonstrated that the pattern of oxidation of one-carbon compounds can be influenced by the integrity of the mitochondrial structure (8, 9) as well as by the presence of compounds such as glycine, cysteine, homocysteine, semicarbazide, pyruvate, n-alanine, amino-guanidine, NAD+, FAD, or ADP (2, 9, 12). The present investigation was undertaken to delineate some of the pathways of interconversion of the oxidized N-methyl group between the levels of formaldehyde, formate, and carbon dioxide.
in part by NIH Grant AM 14315. A report of this work has been presented address: Department of Biochemistry, Medicine, East Carolina University, North Carolina 27834. 443
0003-Y861/78/1852-0443$02.00/O
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved
444
LEWIS MATERIALS
AND
METHODS
Methods. Mitochondria were isolated as described previously and were washed and finally suspended in either phosphate or pyrophosphate (9). Protein was determined by the method of Lowry et al. (13). Oxygen consumption was measured manometrically in a Gilson differential respirometer as described in the table legends. H”CH0 was analyzed by procedures employed earlier (6). [14C]Formate was measured by the method of Weinhouse and Friedman (14). [“ClCarbon dioxide was determined by a modification of the procedure of Yardley (15) and absorbing the respiratory CO, in 0.2 ml of 10% KOH in the center well of the respirometer flask. At the end of the incubation period, the K&O, solution was quantitatively transferred to a scintillation vial containing 15 ml of “Aquasol” (New England Nuclear) and the samples were counted until the counting rate became constant and there was no further evidence of the alkali-induced chemiluminescense which is observed initially in this system. The validity of this procedure was established using standard Na,‘%O,, purchased from New England Nuclear. The tetrahydrofolate derivatives were separated and identified chromatographically as described by Kaufman et al. (16). Substrates and coenzymes. All of the following were obtained from New England Nuclear: [“CHJsarcosine, H”CH0, H’%OOH, S-ll%H,ladenosylmethionine, [l-‘4Clglycine, [Z-‘4Clglycine, [U-‘*C]serine, and [5,10-14CH,]FH,.3 NAD+ and NADP+ were obtained from Sigma Chemical Co. Sarcosine (Fluka AB) was recrystallized from ethanol-water. RESULTS
AND
DISCUSSION
One-carbon oxidation in phosphatetreated mitochondria. In mitochondria swollen with phosphate, the methyl carbon of sarcosine can be oxidized beyond the level of formaldehyde upon the addition of NAD+ to the system (2). The accumulation of serine is not affected significantly and the increased oxygen uptake corresponds to the decrease in formaldehyde and the appearance of formate (2). The present studies demonstrated that formate accumulation is enhanced significantly when NADP+ is added to the 3 Abbreviations used: NS,N”‘-methylenetetrahydrofolate, 5,10-CH,-FH,; N”,N”‘-methenyltetrahydrofolate, 5,10-methenyl FHI; N”‘-formyltetrahydrofolate, IO-formyl FH,; S-adenosylmethionine, SAM.
ET AL.
NAD+-supplemented mitochondria (Table I). The increased oxygen consumption in the presence of the combined pyridine nucleotides corresponds to the decrease in the accumulation of formaldehyde and serine. The production of carbon dioxide under these conditions is undetectable. These results demonstrate that formate can be derived from the methyl carbon of sarcosine by both NAD+- and NADP+-dependent reactions. The present studies also showed that exogenous formaldehyde is not oxidized in intact, phosphate-treated mitochondria supplemented with NADP+ alone, under conditions in which NAD+ stimulates oxidation. Consistent with this observation, a formaldehyde dehydrogenase activity specific for NAD+ can be detected in the soluble fraction of mitochondria broken by sonic irradiation or lysis.4 It may be assumed that this NAD+-dependent dehydrogenase is functional in the oxidation of the “ordinary” formaldehyde derived from sarcosine in the phosphate-washed mitochondria. On the other hand, the most likely NADP+-dependent enzyme which could account for the concurrent increase in accumulation of formate and decrease in serine levels is 5,10CH,-FH, dehydrogenase (17). Confirming earlier findings (21, the studies reported here showed that exogenous glycine causes a marked increase in serine formation and a corresponding decrease in the accumulation of formaldehyde. In addition, it was found that the formation of formate in the absence of exogenous cofactors is not significant when sarcosine is oxidized in the presence of glycine. If the system is supplemented with only NAD+, there is an increase in the amount of formate which is greater than the quantity produced in the presence of NADP+ alone. As in the other systems described in the present study, the addition of NADP+ alone causes an increase in the accumulation of formaldehyde and a decrease in serine formation. When both NAD+ and NADP+ are introduced, formate production becomes maximal and the amounts of both serine and formaldehyde isolated are decreased. ’ W. R. Frisell,
unpublished
observations.
MITOCHONDRIAL
OXIDATION
OF
ONE-CARBON
can inhibit 5,10-CH,-FH, dehydrogenase (18). It is interesting, however, that omission of exogenous Mg”+ from the phosphate-buffer system does not affect the accumulation of formaldehyde, formate, and serine. One-carbon oxidation in pyrophosphatetreated mitochondria. Mitochondria isolated in isotonic KC1 and exposed to a reaction medium of 0.075 M potassium pyrophosphate, pH 7.5, exhibit respiratory control (9). In the present studies, it was found that the oxidation of the N-CH, carbon in this system without added cofactors proceeds beyond the level of formaldehyde (Table II). Formate and carbon dioxide account for more than half of the oxidized methyl carbon and the remainder is recovered as serine. This amino acid is produced in higher quantities than in phosphate-swollen mitochondria, and
The distinction between the NAD+- and NADP+-dependent pathways for formate synthesis was also apparent when sarcosine was oxidized in phosphate-swollen mitochondria in the presence of EDTA. As seen in the data of Table I, the accumulation of formaldehyde is decreased more than fourfold in the EDTA-treated mitochondria, whereas serine production is elevated. In the presence of EDTA, formate is a major product of the oxidized methyl carbon and the amount isolated is not affected markedly by the addition of either NAD+ or NADP+ alone. However, when the system is supplemented with the combined nucleotides, 1.5 times as much formate accumulates and carbon dioxide also appears at a significant level. This marked influence of EDTA could result in part from the chelation of intramitochondrial Mg’+ and Ca’+, both of which TABLE EFFECT
OF EXOGENOUS CARBON
Additions
None NAD NADP NAD + NADP
PYRIDINE
NUCLEOTIDES
TO FORMATE
AND
Oxygen uptake (&atoms)
I
EDTA
AND
CARBON
445
COMPOUNDS
DIOXIDE
ON THE OXIDATION
OF THE
IN PHOSPHATE-SWOLLEN
HCHO (pm011
Serine (pmol)
SARCOSINE
METHYL
MITOCHONDRIA~
HCOOH (pmol)
co, (prnol)
-EDTA
+EDTA
-EDTA
+EDTA
-EDTA
+EDTA
-EDTA
+EDTA
-EDTA
+EDTA
8.1 11.8 9.7 16.4
14.6 14.5 17.9 18.6
6.18 4.06 7.39 2.18
1.39 1.52 1.56 0.16
2.60 1.95 0.67 0.40
3.97 3.31 2.54 1.46
0.34 3.14 0.75 6.00
2.64 3.07 3.26 5.92
0 0 0 0
0.62 0.79 1.25 1.01
n Reaction components, each in 0.075 M potassium phosphate-O.0001 M MgCI, or 0.075 M phosphate0.001 M EDTA, pH 7.8: mitochondria, 17.2 mg of protein, 1.5 ml; [‘4CH,]sarcosine, 10 gmol (1.2 x lo4 cpm/ pmol, corrected); NAD’, 2.6 pmol; NADP’, 2.4 pmol; same buffer, as required, to final volume of 2.6 ml. Temperature, 30°C. 0, measured to cessation of uptake (140 min). TABLE OXIDATION
Additions
None NAD NADP NAD + NADP
OF THE
SARCOSINE
METHYL
Oxygen uptake (patoms)
CARBON
IN
HCHO (pm4
II MITOCHONDRIA
Serine (pmol)
TREATED
WITH
PYROPHOSPHATE”
HCOOH ( pmol)
co, (~rnol)
-Mg
+Mg
-Mg
+Mg
-Mg
+Mg
-Mg
+Mg
-Mg
+Mg
17.6 16.4 19.5 22.8
11.5 10.6 12.8 17.7
0.01 0.12 0.03 0.02
1.90 2.00 1.78 0.28
3.46 3.62 2.93 1.99
4.19 4.00 3.35 2.53
3.02 2.72 3.18 3.00
1.33 1.15 1.63 3.06
2.57 2.07 3.03 4.04
1.25 1.09 1.80 2.65
n Reaction components, each in 0.075 M potassium pyrophosphate, pH 7.6 2 0.01 M MgCI, as indicated: mitochondria, 25.0 mg of protein, in 1.0 ml of 0.15 M KCl; [‘4CH,]sarcosine, 10 pmol (1.1 x lo* cpm/~mol, corrected); NAD+, 2.6 Fmol; NADP+, 2.4 pmol; buffer, ? Mg2+, to final volume of 2.6 ml. Temperature, 30°C. 0, uptake measured to completion (140 min).
446
LEWIS
formaldehyde does not accumulate. The amount of formate accumulating in the system is not enhanced by the combined addition of NAD+ and NADP+. However, the amount of carbon dioxide formed is increased significantly and there is a corresponding decrease in the quantity of serine isolated. In contrast to its inability to affect methyl-carbon oxidation in the phosphatetreated mitochondria, exogenous Mg’+ markedly influences the stoichiometry of the reaction products of sarcosine in the pyrophosphate system. Thus, as demonstrated by the data in Table II, formaldehyde becomes a prominent product, and the decrease in the total of formate and carbon dioxide isolated under these conditions is equivalent to the formaldehyde which accumulates. This same pattern of oxidation is obtained when NAD+ and NADP+ are added individually to the Mg2+-supplemented preparations. On the other hand, when both of the pyridine nucleotides are introduced, formaldehyde does not accumulate and formate and carbon dioxide become the major products. In the presence of EDTA (0.001 M), the pattern of oxidation in the pyrophosphatetreated mitochondria is not significantly different from that obtained when exogenous Mg2+ is omitted from the medium. As in the case of the phosphate-swollen mitochondria, exogenous glycine increases the yield of serine-p-C derived from the oxidized sarcosine-methyl and there is a proportionate decrease in the accumulation of formaldehyde, formate, and carbon dioxide. In addition to the N-methyl group of sarcosine, the p-carbon of serine and both carbons of glycine can also be oxidized to carbon dioxide in the pyrophosphatetreated mitochondria (Table III). Except for [lJ4Clglycine, the production of carbon dioxide is enhanced in the presence of NADP+ (+ NAD+) and is suppressed by addition of Mg2+.5 5 In the present experiments, it was observed that the rate of oxidation of glycine plus sarcosine is equal to the sum of the rates obtained when saturation levels of the substrates are oxidized in-
ET AL TABLE OXIDATION
OF SERINE
III
AND
GLYCINE
PYROPHOSPHATE-TREATED
Additions
Micromoles
of CO,
IN
from:
Serine
[2-‘“CJGlycine
[l-“C]Glycine
3.4 3.1 4.5 5.9
1.4 1.3 1.9 2.5
2.7 2.4 2.8 3.1
L-[U-%1-
None NAD NADP NAD + NADP
TO CO,
MITOCHONDRIA”
(1 Reaction conditions same as in Table II except Mg’+ omitted: mitochondria, 20.0 mg of protein, in 1.0 ml of 0.15 M KCI; L-[U-“Clserine, 10 pmol (1.5 x lo4 cpm/pmol, corrected); [2-“Clglycine, 10 wmol (1.7 x 10’ cpm/pmol, corrected); and [l-Ylglycine, 10 pmol (1.2 x lo4 cpm/pmol, corrected).
Methyl-carbon oxidation linked to the methionine:N-methyl glycine transferase system. The pioneering studies of Mackenzie and co-workers (19, 20) established that the methyl group of methionine can be converted to carbon dioxide at a significant rate in mammalian tissues and that the addition of choline to the diet increases the rate of oxidation of methionine substantially (21). Although the enzymology of oxidation of the methyl carbon to carbon dioxide has not yet been delineated, it has been demonstrated that cytoplasmic components are required in addition to the mitochondria for the oxidation of the methionine-methyl (3). It has also been suggested that a cytoplasmic methionine:Nmethylglycine transferase plays a role in this oxidation (4). Similar to earlier results obtained with phosphate-treated mitochondria (71, the present studies showed that the oxidation of methionine in the pyrophosphatetreated mitochondria is not extensive, even in the presence of exogenous glycine. Thus, during an incubation period in which all of the sarcosine-methyl is oxidized, the combined yield of formaldehyde, serine, formate, and carbon dioxide from the methionine-methyl is only 6 to 8%. Similar results are obtained with an unfractionated homogenate. Likewise, when dividually. These results ways are independent.
indicate
that
the two path-
MITOCHONDRIAL TABLE
OXIDATION
OF
IV
EFFECT OF GLYCINE ON OXIDATION OF S[“CHJADENOSYLMETHIONINE IN RAT LIVER HOMOGENATES”
Oxygen uptake (patoms)
-Glycine +Glycine
Recovery of products as percentage of incubated I’4CH,,lSAM HCHO
SerineP-C
HCOOH
CO,
1.3 4.4
1.2 21.5
5.4 11.3
2.9 8.8
14.5 37.2
o The reaction components were: whole homogenate, 30 mg of protein, in 0.25 M sucrose, 1.0 ml; s[‘“CHJadenosylmethionine, 10 pmol (6.0 x lO’cpm/ pmol); and glycine, 20 pmol. Substrates were dissolved in 0.075 M potassium pyrophosphate, pH 7.6 + 0.01 M MgCl, and the same buffers were added to the reaction mixtures to a final volume of 2.6 ml. Temperature, 38°C. Incubation time, 160 min.
S-[‘4CH,ladenosylmethionine is oxidized in the homogenate system, only 10 to 12% of the methyl carbon is recovered as formaldehyde, serine, formate, and carbon dioxide (Table IV). In the presence of glytine, however, the total of these oxidation products is increased fourfold. The accumulation of serine increases almost 20-fold and carbon dioxide production is trebled. These results indicate that the N-methyl glycine transferase system can play a major role in the oxidation of the methionine methyl group via sarcosine. Thus, transmethylation between SAM and glycine may serve not only to regulate cellular levels of S-adenosylmethionine and S-adenosylhomocysteine (4) but can also provide an important source of one-carbon HCHb>
(NM+)
ONE-CARBON
447
intermediates at the oxidation levels of formaldehyde and formate. The nature of “active formaldehyde” and “active formate” derived from oxidative N-demethylation. With the phosphateand pyrophosphate-treated mitochondria employed in the present studies, neither exogenous formaldehyde nor formate was found to be converted to [3-Clserine or carbon dioxide under conditions in which [14CH,lsarcosine, l’4CH:,JSAM, 12-‘4Clglytine, and [ l-‘4C]glycine gave high yields of formaldehyde, [3-Clserine, formate, or carbon dioxide. These results, together with earlier findings (2, 8), demonstrated that an initial product of the dehydrogenation of the sarcosine-methyl group is very accessible for serine synthesis and/or oxidation to the level of formate and carbon dioxide but cannot equilibrate readily with either exogenous formaldehyde or formate. The origin of “active” and “ordinary” formaldehyde and the sequence of reactions accounting for the oxidative products of these compounds can be formulated as shown in Scheme 1. The reciprocal relationship between serine accumulation and the NADP+-stimulated production of formate and carbon dioxide observed under the conditions of our experiments would be expected if 5,10CH,FH, were a product of the oxidized methyl group. This one-carbon compound, “active formaldehyde,” is a major precursor of the 3-C of serine (22, 23) and can also be oxidized to the Nj,N’O-methenylor N’“-formyl derivatives of FH, by an NADP+-specific dehydrogenase (17, 23,
HCOOH
2H. -i-,H,i>
Ser-3-C SCHEME
COMPOUNDS
1
448
LEWIS
20
30 Fraction
ET AL.
40
50
(2.5ml)
60
No.
FIG. 1. Oxidation of [‘4CH,]sarcosine to N5,N”‘-[‘4C]methylene FH, and N1”-[14C]formyl FH, in pyrophosphate-treated mitochondria. The reaction conditions were the same as described in Table II, except that Mgy+ was omitted from the medium. Flask contents: mitochondria from 2 g of liver, 1.0 ml; 0.075 M pyrophosphate buffer, 1.4 ml; and [‘%H,lsarcosine, 10 pmol (2.3 x IO” cpm/pmol, corrected). Following incubation at 30°C for 10 min, the pH of the reaction mixtures was adjusted to 9.5. The flask contents were heated for 2 min in a boiling water bath and cooled. Following addition of carrier N5,N1”-methenylFH, (0.2 ml) and NS,N’O-CH,-FH, (0.1 ml), the samples were centrifuged at 10,OOOg for 30 min. When required, the supernatant fractions were stored frozen. For chromatographic analysis, a 2.1-ml aliquot was applied to a DEAE-cellulose column (1 x 60 cm). The column was developed with a gradient of 0.004-0.4 M NaHCO, (16). O--O, A,,, “,,,; O-0, A,,, “,,,; A- - - -A, cpm/O.l ml; q - - -0, HCHO (arbitrary units). Elution peaks: Fraction 27, N’“formyl FH,; Fraction 38, N”‘-formyl folate; Fractions 49 and 55, n,L-isomers of JP,N”‘-CH,FH,.
24). The oxidation of N*O-formyl-FH,, a major folate derivative in liver mitochondria (24), to carbon dioxide is also NADP+dependent (25). In a mitochondrial system capable of serine synthesis, the formation of carbon dioxide from the N-methyl carbon of sarcosine, but not from exogenous formaldehyde and formate, suggests that N’O-formyl-FH4 derived from the “active formaldehyde” is the direct precursor of the carbon dioxide. Consistent with this conclusion, [5,10J4CH2]FH4 and [lo“C]formyl FH, were identified as oxidation products of [14CH,lsarcosine in the pyrophosphate-treated mitochondria (Fig. 1). It was also found that in the oxidation of exogenous [5,10-14CH,1FH4 the relative quantities of [3J4Clserine, [14Clformate, and [14C]carbon dioxide isolated were similar to those obtained from the sarcosinemethyl under the same conditions (Table VI. The present studies have extended earlier findings that the oxidation levels at-
TABLE OXIDATION
V OF 5,10-
METWYLENE[‘~C]TETRAHYDROFOLATE IN PYROPHOSPHATE-TREATED MITOCHONDRIA”
Additions None Glycine Sarcosine Glycine + sarcosine
[3-“ClSerine (wol) 0 1.7 0.5 1.6
HL4COOH (pm011 2.5 2.4 1.6 2.9
‘TO, (pm011 0.11
0.07 0.09 0.07
fz Reaction mixture contained: mitochondria, 20.0 mg of protein, in 1.5 ml of 0.15 M KCl; glycine, 40 pmol; sarcosine, 10 pmol; D,L-5,10-‘%H,FH,, 8.55 pmol, 4.16 x lo5 cpm/pmol. All components were dissolved in 0.075 M pyrophosphate buffer, pH 7.6, and the same buffer was added as required to give a total volume of 2.6 ml. Temperature, 30°C. Reaction time, 60 min.
tained by one-carbon compounds in mitochondria are affected by the media to which the preparations are exposed. It is reasonable that the metabolic patterns should be sensitive to changes in morphol-
MITOCHONDRIAL
OXIDATION
ogy, membrane permeability, and efflux of cofactors. In the experiments described here, for example, it was found that the quantity of pyridine nucleotides and folates removed from the mitochondria by washing with pyrophosphate is the same as with phosphate (9). However, only the pyrophosphate-treated preparations retain the ability to undergo the reversible changes between the “orthodox” and “condensed” configurations,6 and only the pyrophosphate-treated mitochondria oxidize the N-methyl carbon to the level of carbon dioxide in the absence of exogenous cofactors. In whatever manner the morphology of the mitochondria and compartmentation of reaction components may influence the oxidative and synthetic reactions of “active formaldehyde” and formaldehyde, these two compounds are not rapidly equilibrated with one another and can, therefore, be distinguished in their metabolic activities. REFERENCES 1. LEWIS, K. F., RANDOLPH, V. M., AND FRISELL, W. R. (1975)Fed. Proc. 34, 590. 2. MACKENZIE, C. G. (1955) in Amino Acid Metabolism (McElroy, W. D., and Glass, B., eds.), pp. 684-726, Johns Hopkins Press, Baltimore, Md. 3. BLUMENSTEIN, JR., AND WILLIAMS, G. R. (1960) Biochem. Biophys. Res. Commun. 3, 259-263. 4. KERR, S. J. (1972) J. Biol. Chem. 247, 42484252. 5. HEADY, J. E., AND KERR, S. J. (1973) J. Biol. Chem. 248, 69-72. 6. MACKENZIE, C. G. (1950) J. Biol. Chem. 186, 351-368. 7. FRISELL, W. R., AND MACKENZIE, C. G. (1961) in Radioactive Isotopes in Physiology, Diagnostics, and Therapy (Schwiegk, H., and Turba, F., eds.), pp. 919-944, Springer-Verlag, Berlin. 6 W. R. Frisell
and I. Bahia,
unpublished
results.
OF
ONE-CARBON
COMPOUNDS
449
W. R.,. AND SORRELL, N. C. (1967) 8. FRISELL, Biochim. Biophys. Acta 131, 207-210. 9. FRISELL, W. R., AND RANDOLPH, V. M. (1973) Biochim. Biophys. Acta 292, 360-365. G. P., AND HUENNEKENS, F. M. (1960) 10. MELL, Fed. Proc. 19, 411. 11. BROTHERS, V., ROWLEY, B. O., AND GERRITSEN (1975) Arch. Biochem. Biophys. 166, 475-482. J. A., CALHOUN, M. C., AND WRISTON, 12. MICHELS, J. C., JR. (1962) Biochim. Biophys. Acta 59, 408-413. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. S., AND FRIEDMAN, B. (1952) J. 14. WEINHOUSE, Biol. Chem. 197, 733-740. H. J. (1964) Nature (London) 240, 15. YARDLEY, 281. B. T., DONALDSON, K. O., AND KER16. KAUFMAN, ESZTESY, J. C. (1963) J. Biol. Chem. 238,14981500. 17. HATEFI, Y., OSBORN, M. J., KAY, L. D., AND HUENNEKENS, F. M. (1957) J. Biol. Chem. 227, 637-647. R. L. (1969) in Frontiers of Biology 18. BLAKLEY, (Neuberger, A., and Tatum, E. L., eds.), Vol. 13, p. 191, Wiley, New York. C. G., CHANDLER, J. P., KELLER, 19. MACKENZIE, E. B., RACHELE, J. R., CROSS, N., MELVILLE, D. B., AND DU VIGNEAUD, V. (1947) J. Biol. Chem. 169, 757-758. 20. MACKENZIE, C. G., RACHELE, J. R., CROSS, N., CHANDLER, J. P., AND DU VIGNEAUD, V. (1950) J. Biol. Chem. 183, 617-626. 21. MACKENZIE, C. G., AND DU VIGNEAUD, V. (1952) J. Biol. Chem. 195, 487-491. 22. GREENBERG, G. R. AND JAENICKE, L., (1957) in Ciba Foundation Symposium on the Chemistry and Biology of Purines (Wolstenholme, G. E. W., and O’Conner, C. M., eds.), p. 204, Little, Brown, Boston, Mass, 23. MATHEWS, C. K., AND HUENNEKENS, F. M. (1960) J. Biol. Chem. 235, 3304-3308. 24. WANG, F. K., KOCH, J., AND STOKSTAD, E. L. R. (1967) Biochem. 2. 346, 458-466. 25 KUTZBACH, C., AND STOKSTAD, E. L. R. (1968) Biochem. Biophys. Res. Commun. 30,111-117.
Oxidation KATHERINE
New
Jersey
Medical
of One-Carbon Compounds to Formate Dioxide in Rat Liver Mitochondria’ F. LEWIS,
School,
College Received
VERRELL WILHELM of Medicine
May
M. RANDOLPH, R. FRISELL’ and Dentistry
27, 1977; revised
of New
September
and Carbon
EDITH
Jerse.y,
NEMETH,
Newark,
New
Jersey
AND
07103
19, 1977
In rat liver mitochondria, swollen with phosphate and supplemented with NAD+, the oxidation of the methyl carbon of sarcosine to formate is enhanced by the addition of NADP+. No carbon dioxide is formed. Formaldehyde and serine, which are the only oxidation products of the methyl group in the absence of the pyridine nucleotides, are decreased by an amount equal to the formate produced. Carbon dioxide, as well as formate, is produced when the mitochondria are treated with EDTA, even without the addition of the pyridine nucleotides. When the mitochondria are exposed to pyrophosphate without added NAD+ and/or NADP+, all of the oxidized sarcosine-methyl can be recovered as formate, [3-Clserine, and carbon dioxide. Formaldehyde accumulates only if the system is supplemented with Mg’+. In the presence of NADP+ or the combined pyridine nucleotides, serine accumulation is depressed by an amount equal to the increase in carbon dioxide production. Both carbons of glycine and the 3-C of serine can also be oxidized to carbon dioxide in the pyrophosphate-treated mitochondria. The oxidation of the methyl carbon of S-adenosylmethionine to formaldehyde, [3-Clserine, formate, and carbon dioxide requires a whole homogenate supplemented with glycine. Neither exogenous formaldehyde nor formate is oxidized to carbon dioxide in any of the mitochondrial systems capable of converting sarcosine-methyl to carbon dioxide. Under conditions in which [NS,N”‘-‘4C-methyleneIand [1L”“-‘4C-formy11tetrahydrofolate can be isolated as intermediate products of [‘4CHnlsarcosine, exogenous lm,A”“-‘4Cmelh.y1erLeltetrahydrofolate can also be converted to [3-“Clserine, [“Clformate, and [‘Xlcarbon dioxide.
In mammalian systems, N-methylglytine (sarcosine) is produced both via the “one-carbon cycle” (2) and by transmethylation between S-adenosylmethionine and glycine (3-5). The N-methyl group is very labile to oxidation (6) and becomes an important precursor for “one-carbon transfer” reactions (7). The three dehydrogenation reactions which produce formaldehyde, formate, and carbon dioxide from the methyl carbon occur exclusively in mitochondria (2, 8, 9). Under certain conditions, the oxidation of the methyl carbon 1 Supported preliminary (1). s Present School of Greenville,
may be restricted to the level of “active formaldehyde” so that the only products are serine and/or formaldehyde (2). However, the implication of folate derivatives in these reactions has not been established unequivocally (10, 11). Subsequent studies have demonstrated that the pattern of oxidation of one-carbon compounds can be influenced by the integrity of the mitochondrial structure (8, 9) as well as by the presence of compounds such as glycine, cysteine, homocysteine, semicarbazide, pyruvate, n-alanine, amino-guanidine, NAD+, FAD, or ADP (2, 9, 12). The present investigation was undertaken to delineate some of the pathways of interconversion of the oxidized N-methyl group between the levels of formaldehyde, formate, and carbon dioxide.
in part by NIH Grant AM 14315. A report of this work has been presented address: Department of Biochemistry, Medicine, East Carolina University, North Carolina 27834. 443
0003-Y861/78/1852-0443$02.00/O
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved
444
LEWIS MATERIALS
AND
METHODS
Methods. Mitochondria were isolated as described previously and were washed and finally suspended in either phosphate or pyrophosphate (9). Protein was determined by the method of Lowry et al. (13). Oxygen consumption was measured manometrically in a Gilson differential respirometer as described in the table legends. H”CH0 was analyzed by procedures employed earlier (6). [14C]Formate was measured by the method of Weinhouse and Friedman (14). [“ClCarbon dioxide was determined by a modification of the procedure of Yardley (15) and absorbing the respiratory CO, in 0.2 ml of 10% KOH in the center well of the respirometer flask. At the end of the incubation period, the K&O, solution was quantitatively transferred to a scintillation vial containing 15 ml of “Aquasol” (New England Nuclear) and the samples were counted until the counting rate became constant and there was no further evidence of the alkali-induced chemiluminescense which is observed initially in this system. The validity of this procedure was established using standard Na,‘%O,, purchased from New England Nuclear. The tetrahydrofolate derivatives were separated and identified chromatographically as described by Kaufman et al. (16). Substrates and coenzymes. All of the following were obtained from New England Nuclear: [“CHJsarcosine, H”CH0, H’%OOH, S-ll%H,ladenosylmethionine, [l-‘4Clglycine, [Z-‘4Clglycine, [U-‘*C]serine, and [5,10-14CH,]FH,.3 NAD+ and NADP+ were obtained from Sigma Chemical Co. Sarcosine (Fluka AB) was recrystallized from ethanol-water. RESULTS
AND
DISCUSSION
One-carbon oxidation in phosphatetreated mitochondria. In mitochondria swollen with phosphate, the methyl carbon of sarcosine can be oxidized beyond the level of formaldehyde upon the addition of NAD+ to the system (2). The accumulation of serine is not affected significantly and the increased oxygen uptake corresponds to the decrease in formaldehyde and the appearance of formate (2). The present studies demonstrated that formate accumulation is enhanced significantly when NADP+ is added to the 3 Abbreviations used: NS,N”‘-methylenetetrahydrofolate, 5,10-CH,-FH,; N”,N”‘-methenyltetrahydrofolate, 5,10-methenyl FHI; N”‘-formyltetrahydrofolate, IO-formyl FH,; S-adenosylmethionine, SAM.
ET AL.
NAD+-supplemented mitochondria (Table I). The increased oxygen consumption in the presence of the combined pyridine nucleotides corresponds to the decrease in the accumulation of formaldehyde and serine. The production of carbon dioxide under these conditions is undetectable. These results demonstrate that formate can be derived from the methyl carbon of sarcosine by both NAD+- and NADP+-dependent reactions. The present studies also showed that exogenous formaldehyde is not oxidized in intact, phosphate-treated mitochondria supplemented with NADP+ alone, under conditions in which NAD+ stimulates oxidation. Consistent with this observation, a formaldehyde dehydrogenase activity specific for NAD+ can be detected in the soluble fraction of mitochondria broken by sonic irradiation or lysis.4 It may be assumed that this NAD+-dependent dehydrogenase is functional in the oxidation of the “ordinary” formaldehyde derived from sarcosine in the phosphate-washed mitochondria. On the other hand, the most likely NADP+-dependent enzyme which could account for the concurrent increase in accumulation of formate and decrease in serine levels is 5,10CH,-FH, dehydrogenase (17). Confirming earlier findings (21, the studies reported here showed that exogenous glycine causes a marked increase in serine formation and a corresponding decrease in the accumulation of formaldehyde. In addition, it was found that the formation of formate in the absence of exogenous cofactors is not significant when sarcosine is oxidized in the presence of glycine. If the system is supplemented with only NAD+, there is an increase in the amount of formate which is greater than the quantity produced in the presence of NADP+ alone. As in the other systems described in the present study, the addition of NADP+ alone causes an increase in the accumulation of formaldehyde and a decrease in serine formation. When both NAD+ and NADP+ are introduced, formate production becomes maximal and the amounts of both serine and formaldehyde isolated are decreased. ’ W. R. Frisell,
unpublished
observations.
MITOCHONDRIAL
OXIDATION
OF
ONE-CARBON
can inhibit 5,10-CH,-FH, dehydrogenase (18). It is interesting, however, that omission of exogenous Mg”+ from the phosphate-buffer system does not affect the accumulation of formaldehyde, formate, and serine. One-carbon oxidation in pyrophosphatetreated mitochondria. Mitochondria isolated in isotonic KC1 and exposed to a reaction medium of 0.075 M potassium pyrophosphate, pH 7.5, exhibit respiratory control (9). In the present studies, it was found that the oxidation of the N-CH, carbon in this system without added cofactors proceeds beyond the level of formaldehyde (Table II). Formate and carbon dioxide account for more than half of the oxidized methyl carbon and the remainder is recovered as serine. This amino acid is produced in higher quantities than in phosphate-swollen mitochondria, and
The distinction between the NAD+- and NADP+-dependent pathways for formate synthesis was also apparent when sarcosine was oxidized in phosphate-swollen mitochondria in the presence of EDTA. As seen in the data of Table I, the accumulation of formaldehyde is decreased more than fourfold in the EDTA-treated mitochondria, whereas serine production is elevated. In the presence of EDTA, formate is a major product of the oxidized methyl carbon and the amount isolated is not affected markedly by the addition of either NAD+ or NADP+ alone. However, when the system is supplemented with the combined nucleotides, 1.5 times as much formate accumulates and carbon dioxide also appears at a significant level. This marked influence of EDTA could result in part from the chelation of intramitochondrial Mg’+ and Ca’+, both of which TABLE EFFECT
OF EXOGENOUS CARBON
Additions
None NAD NADP NAD + NADP
PYRIDINE
NUCLEOTIDES
TO FORMATE
AND
Oxygen uptake (&atoms)
I
EDTA
AND
CARBON
445
COMPOUNDS
DIOXIDE
ON THE OXIDATION
OF THE
IN PHOSPHATE-SWOLLEN
HCHO (pm011
Serine (pmol)
SARCOSINE
METHYL
MITOCHONDRIA~
HCOOH (pmol)
co, (prnol)
-EDTA
+EDTA
-EDTA
+EDTA
-EDTA
+EDTA
-EDTA
+EDTA
-EDTA
+EDTA
8.1 11.8 9.7 16.4
14.6 14.5 17.9 18.6
6.18 4.06 7.39 2.18
1.39 1.52 1.56 0.16
2.60 1.95 0.67 0.40
3.97 3.31 2.54 1.46
0.34 3.14 0.75 6.00
2.64 3.07 3.26 5.92
0 0 0 0
0.62 0.79 1.25 1.01
n Reaction components, each in 0.075 M potassium phosphate-O.0001 M MgCI, or 0.075 M phosphate0.001 M EDTA, pH 7.8: mitochondria, 17.2 mg of protein, 1.5 ml; [‘4CH,]sarcosine, 10 gmol (1.2 x lo4 cpm/ pmol, corrected); NAD’, 2.6 pmol; NADP’, 2.4 pmol; same buffer, as required, to final volume of 2.6 ml. Temperature, 30°C. 0, measured to cessation of uptake (140 min). TABLE OXIDATION
Additions
None NAD NADP NAD + NADP
OF THE
SARCOSINE
METHYL
Oxygen uptake (patoms)
CARBON
IN
HCHO (pm4
II MITOCHONDRIA
Serine (pmol)
TREATED
WITH
PYROPHOSPHATE”
HCOOH ( pmol)
co, (~rnol)
-Mg
+Mg
-Mg
+Mg
-Mg
+Mg
-Mg
+Mg
-Mg
+Mg
17.6 16.4 19.5 22.8
11.5 10.6 12.8 17.7
0.01 0.12 0.03 0.02
1.90 2.00 1.78 0.28
3.46 3.62 2.93 1.99
4.19 4.00 3.35 2.53
3.02 2.72 3.18 3.00
1.33 1.15 1.63 3.06
2.57 2.07 3.03 4.04
1.25 1.09 1.80 2.65
n Reaction components, each in 0.075 M potassium pyrophosphate, pH 7.6 2 0.01 M MgCI, as indicated: mitochondria, 25.0 mg of protein, in 1.0 ml of 0.15 M KCl; [‘4CH,]sarcosine, 10 pmol (1.1 x lo* cpm/~mol, corrected); NAD+, 2.6 Fmol; NADP+, 2.4 pmol; buffer, ? Mg2+, to final volume of 2.6 ml. Temperature, 30°C. 0, uptake measured to completion (140 min).
446
LEWIS
formaldehyde does not accumulate. The amount of formate accumulating in the system is not enhanced by the combined addition of NAD+ and NADP+. However, the amount of carbon dioxide formed is increased significantly and there is a corresponding decrease in the quantity of serine isolated. In contrast to its inability to affect methyl-carbon oxidation in the phosphatetreated mitochondria, exogenous Mg’+ markedly influences the stoichiometry of the reaction products of sarcosine in the pyrophosphate system. Thus, as demonstrated by the data in Table II, formaldehyde becomes a prominent product, and the decrease in the total of formate and carbon dioxide isolated under these conditions is equivalent to the formaldehyde which accumulates. This same pattern of oxidation is obtained when NAD+ and NADP+ are added individually to the Mg2+-supplemented preparations. On the other hand, when both of the pyridine nucleotides are introduced, formaldehyde does not accumulate and formate and carbon dioxide become the major products. In the presence of EDTA (0.001 M), the pattern of oxidation in the pyrophosphatetreated mitochondria is not significantly different from that obtained when exogenous Mg2+ is omitted from the medium. As in the case of the phosphate-swollen mitochondria, exogenous glycine increases the yield of serine-p-C derived from the oxidized sarcosine-methyl and there is a proportionate decrease in the accumulation of formaldehyde, formate, and carbon dioxide. In addition to the N-methyl group of sarcosine, the p-carbon of serine and both carbons of glycine can also be oxidized to carbon dioxide in the pyrophosphatetreated mitochondria (Table III). Except for [lJ4Clglycine, the production of carbon dioxide is enhanced in the presence of NADP+ (+ NAD+) and is suppressed by addition of Mg2+.5 5 In the present experiments, it was observed that the rate of oxidation of glycine plus sarcosine is equal to the sum of the rates obtained when saturation levels of the substrates are oxidized in-
ET AL TABLE OXIDATION
OF SERINE
III
AND
GLYCINE
PYROPHOSPHATE-TREATED
Additions
Micromoles
of CO,
IN
from:
Serine
[2-‘“CJGlycine
[l-“C]Glycine
3.4 3.1 4.5 5.9
1.4 1.3 1.9 2.5
2.7 2.4 2.8 3.1
L-[U-%1-
None NAD NADP NAD + NADP
TO CO,
MITOCHONDRIA”
(1 Reaction conditions same as in Table II except Mg’+ omitted: mitochondria, 20.0 mg of protein, in 1.0 ml of 0.15 M KCI; L-[U-“Clserine, 10 pmol (1.5 x lo4 cpm/pmol, corrected); [2-“Clglycine, 10 wmol (1.7 x 10’ cpm/pmol, corrected); and [l-Ylglycine, 10 pmol (1.2 x lo4 cpm/pmol, corrected).
Methyl-carbon oxidation linked to the methionine:N-methyl glycine transferase system. The pioneering studies of Mackenzie and co-workers (19, 20) established that the methyl group of methionine can be converted to carbon dioxide at a significant rate in mammalian tissues and that the addition of choline to the diet increases the rate of oxidation of methionine substantially (21). Although the enzymology of oxidation of the methyl carbon to carbon dioxide has not yet been delineated, it has been demonstrated that cytoplasmic components are required in addition to the mitochondria for the oxidation of the methionine-methyl (3). It has also been suggested that a cytoplasmic methionine:Nmethylglycine transferase plays a role in this oxidation (4). Similar to earlier results obtained with phosphate-treated mitochondria (71, the present studies showed that the oxidation of methionine in the pyrophosphatetreated mitochondria is not extensive, even in the presence of exogenous glycine. Thus, during an incubation period in which all of the sarcosine-methyl is oxidized, the combined yield of formaldehyde, serine, formate, and carbon dioxide from the methionine-methyl is only 6 to 8%. Similar results are obtained with an unfractionated homogenate. Likewise, when dividually. These results ways are independent.
indicate
that
the two path-
MITOCHONDRIAL TABLE
OXIDATION
OF
IV
EFFECT OF GLYCINE ON OXIDATION OF S[“CHJADENOSYLMETHIONINE IN RAT LIVER HOMOGENATES”
Oxygen uptake (patoms)
-Glycine +Glycine
Recovery of products as percentage of incubated I’4CH,,lSAM HCHO
SerineP-C
HCOOH
CO,
1.3 4.4
1.2 21.5
5.4 11.3
2.9 8.8
14.5 37.2
o The reaction components were: whole homogenate, 30 mg of protein, in 0.25 M sucrose, 1.0 ml; s[‘“CHJadenosylmethionine, 10 pmol (6.0 x lO’cpm/ pmol); and glycine, 20 pmol. Substrates were dissolved in 0.075 M potassium pyrophosphate, pH 7.6 + 0.01 M MgCl, and the same buffers were added to the reaction mixtures to a final volume of 2.6 ml. Temperature, 38°C. Incubation time, 160 min.
S-[‘4CH,ladenosylmethionine is oxidized in the homogenate system, only 10 to 12% of the methyl carbon is recovered as formaldehyde, serine, formate, and carbon dioxide (Table IV). In the presence of glytine, however, the total of these oxidation products is increased fourfold. The accumulation of serine increases almost 20-fold and carbon dioxide production is trebled. These results indicate that the N-methyl glycine transferase system can play a major role in the oxidation of the methionine methyl group via sarcosine. Thus, transmethylation between SAM and glycine may serve not only to regulate cellular levels of S-adenosylmethionine and S-adenosylhomocysteine (4) but can also provide an important source of one-carbon HCHb>
(NM+)
ONE-CARBON
447
intermediates at the oxidation levels of formaldehyde and formate. The nature of “active formaldehyde” and “active formate” derived from oxidative N-demethylation. With the phosphateand pyrophosphate-treated mitochondria employed in the present studies, neither exogenous formaldehyde nor formate was found to be converted to [3-Clserine or carbon dioxide under conditions in which [14CH,lsarcosine, l’4CH:,JSAM, 12-‘4Clglytine, and [ l-‘4C]glycine gave high yields of formaldehyde, [3-Clserine, formate, or carbon dioxide. These results, together with earlier findings (2, 8), demonstrated that an initial product of the dehydrogenation of the sarcosine-methyl group is very accessible for serine synthesis and/or oxidation to the level of formate and carbon dioxide but cannot equilibrate readily with either exogenous formaldehyde or formate. The origin of “active” and “ordinary” formaldehyde and the sequence of reactions accounting for the oxidative products of these compounds can be formulated as shown in Scheme 1. The reciprocal relationship between serine accumulation and the NADP+-stimulated production of formate and carbon dioxide observed under the conditions of our experiments would be expected if 5,10CH,FH, were a product of the oxidized methyl group. This one-carbon compound, “active formaldehyde,” is a major precursor of the 3-C of serine (22, 23) and can also be oxidized to the Nj,N’O-methenylor N’“-formyl derivatives of FH, by an NADP+-specific dehydrogenase (17, 23,
HCOOH
2H. -i-,H,i>
Ser-3-C SCHEME
COMPOUNDS
1
448
LEWIS
20
30 Fraction
ET AL.
40
50
(2.5ml)
60
No.
FIG. 1. Oxidation of [‘4CH,]sarcosine to N5,N”‘-[‘4C]methylene FH, and N1”-[14C]formyl FH, in pyrophosphate-treated mitochondria. The reaction conditions were the same as described in Table II, except that Mgy+ was omitted from the medium. Flask contents: mitochondria from 2 g of liver, 1.0 ml; 0.075 M pyrophosphate buffer, 1.4 ml; and [‘%H,lsarcosine, 10 pmol (2.3 x IO” cpm/pmol, corrected). Following incubation at 30°C for 10 min, the pH of the reaction mixtures was adjusted to 9.5. The flask contents were heated for 2 min in a boiling water bath and cooled. Following addition of carrier N5,N1”-methenylFH, (0.2 ml) and NS,N’O-CH,-FH, (0.1 ml), the samples were centrifuged at 10,OOOg for 30 min. When required, the supernatant fractions were stored frozen. For chromatographic analysis, a 2.1-ml aliquot was applied to a DEAE-cellulose column (1 x 60 cm). The column was developed with a gradient of 0.004-0.4 M NaHCO, (16). O--O, A,,, “,,,; O-0, A,,, “,,,; A- - - -A, cpm/O.l ml; q - - -0, HCHO (arbitrary units). Elution peaks: Fraction 27, N’“formyl FH,; Fraction 38, N”‘-formyl folate; Fractions 49 and 55, n,L-isomers of JP,N”‘-CH,FH,.
24). The oxidation of N*O-formyl-FH,, a major folate derivative in liver mitochondria (24), to carbon dioxide is also NADP+dependent (25). In a mitochondrial system capable of serine synthesis, the formation of carbon dioxide from the N-methyl carbon of sarcosine, but not from exogenous formaldehyde and formate, suggests that N’O-formyl-FH4 derived from the “active formaldehyde” is the direct precursor of the carbon dioxide. Consistent with this conclusion, [5,10J4CH2]FH4 and [lo“C]formyl FH, were identified as oxidation products of [14CH,lsarcosine in the pyrophosphate-treated mitochondria (Fig. 1). It was also found that in the oxidation of exogenous [5,10-14CH,1FH4 the relative quantities of [3J4Clserine, [14Clformate, and [14C]carbon dioxide isolated were similar to those obtained from the sarcosinemethyl under the same conditions (Table VI. The present studies have extended earlier findings that the oxidation levels at-
TABLE OXIDATION
V OF 5,10-
METWYLENE[‘~C]TETRAHYDROFOLATE IN PYROPHOSPHATE-TREATED MITOCHONDRIA”
Additions None Glycine Sarcosine Glycine + sarcosine
[3-“ClSerine (wol) 0 1.7 0.5 1.6
HL4COOH (pm011 2.5 2.4 1.6 2.9
‘TO, (pm011 0.11
0.07 0.09 0.07
fz Reaction mixture contained: mitochondria, 20.0 mg of protein, in 1.5 ml of 0.15 M KCl; glycine, 40 pmol; sarcosine, 10 pmol; D,L-5,10-‘%H,FH,, 8.55 pmol, 4.16 x lo5 cpm/pmol. All components were dissolved in 0.075 M pyrophosphate buffer, pH 7.6, and the same buffer was added as required to give a total volume of 2.6 ml. Temperature, 30°C. Reaction time, 60 min.
tained by one-carbon compounds in mitochondria are affected by the media to which the preparations are exposed. It is reasonable that the metabolic patterns should be sensitive to changes in morphol-
MITOCHONDRIAL
OXIDATION
ogy, membrane permeability, and efflux of cofactors. In the experiments described here, for example, it was found that the quantity of pyridine nucleotides and folates removed from the mitochondria by washing with pyrophosphate is the same as with phosphate (9). However, only the pyrophosphate-treated preparations retain the ability to undergo the reversible changes between the “orthodox” and “condensed” configurations,6 and only the pyrophosphate-treated mitochondria oxidize the N-methyl carbon to the level of carbon dioxide in the absence of exogenous cofactors. In whatever manner the morphology of the mitochondria and compartmentation of reaction components may influence the oxidative and synthetic reactions of “active formaldehyde” and formaldehyde, these two compounds are not rapidly equilibrated with one another and can, therefore, be distinguished in their metabolic activities. REFERENCES 1. LEWIS, K. F., RANDOLPH, V. M., AND FRISELL, W. R. (1975)Fed. Proc. 34, 590. 2. MACKENZIE, C. G. (1955) in Amino Acid Metabolism (McElroy, W. D., and Glass, B., eds.), pp. 684-726, Johns Hopkins Press, Baltimore, Md. 3. BLUMENSTEIN, JR., AND WILLIAMS, G. R. (1960) Biochem. Biophys. Res. Commun. 3, 259-263. 4. KERR, S. J. (1972) J. Biol. Chem. 247, 42484252. 5. HEADY, J. E., AND KERR, S. J. (1973) J. Biol. Chem. 248, 69-72. 6. MACKENZIE, C. G. (1950) J. Biol. Chem. 186, 351-368. 7. FRISELL, W. R., AND MACKENZIE, C. G. (1961) in Radioactive Isotopes in Physiology, Diagnostics, and Therapy (Schwiegk, H., and Turba, F., eds.), pp. 919-944, Springer-Verlag, Berlin. 6 W. R. Frisell
and I. Bahia,
unpublished
results.
OF
ONE-CARBON
COMPOUNDS
449
W. R.,. AND SORRELL, N. C. (1967) 8. FRISELL, Biochim. Biophys. Acta 131, 207-210. 9. FRISELL, W. R., AND RANDOLPH, V. M. (1973) Biochim. Biophys. Acta 292, 360-365. G. P., AND HUENNEKENS, F. M. (1960) 10. MELL, Fed. Proc. 19, 411. 11. BROTHERS, V., ROWLEY, B. O., AND GERRITSEN (1975) Arch. Biochem. Biophys. 166, 475-482. J. A., CALHOUN, M. C., AND WRISTON, 12. MICHELS, J. C., JR. (1962) Biochim. Biophys. Acta 59, 408-413. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. S., AND FRIEDMAN, B. (1952) J. 14. WEINHOUSE, Biol. Chem. 197, 733-740. H. J. (1964) Nature (London) 240, 15. YARDLEY, 281. B. T., DONALDSON, K. O., AND KER16. KAUFMAN, ESZTESY, J. C. (1963) J. Biol. Chem. 238,14981500. 17. HATEFI, Y., OSBORN, M. J., KAY, L. D., AND HUENNEKENS, F. M. (1957) J. Biol. Chem. 227, 637-647. R. L. (1969) in Frontiers of Biology 18. BLAKLEY, (Neuberger, A., and Tatum, E. L., eds.), Vol. 13, p. 191, Wiley, New York. C. G., CHANDLER, J. P., KELLER, 19. MACKENZIE, E. B., RACHELE, J. R., CROSS, N., MELVILLE, D. B., AND DU VIGNEAUD, V. (1947) J. Biol. Chem. 169, 757-758. 20. MACKENZIE, C. G., RACHELE, J. R., CROSS, N., CHANDLER, J. P., AND DU VIGNEAUD, V. (1950) J. Biol. Chem. 183, 617-626. 21. MACKENZIE, C. G., AND DU VIGNEAUD, V. (1952) J. Biol. Chem. 195, 487-491. 22. GREENBERG, G. R. AND JAENICKE, L., (1957) in Ciba Foundation Symposium on the Chemistry and Biology of Purines (Wolstenholme, G. E. W., and O’Conner, C. M., eds.), p. 204, Little, Brown, Boston, Mass, 23. MATHEWS, C. K., AND HUENNEKENS, F. M. (1960) J. Biol. Chem. 235, 3304-3308. 24. WANG, F. K., KOCH, J., AND STOKSTAD, E. L. R. (1967) Biochem. 2. 346, 458-466. 25 KUTZBACH, C., AND STOKSTAD, E. L. R. (1968) Biochem. Biophys. Res. Commun. 30,111-117.
