55 1
ClinicalScience (1991) 81,551-556
Betaine-homocysteine methyltransferase: organ distribution in man, pig and rat and subcellular distribution in the rat M. P. McKEEVER, D. G. WEIR*, A. MOLLOY*
AND
J. M. SCOTT
Departments of Biochemistry and *Clinical Medicine, Trinity College, Dublin, Republic of Ireland
(Received 28 February/l3 May 1991; accepted 21 May 1991)
SUMMARY 1. Conflicting reports exist as to the organ distribution of betaine-homocysteine methyltransferase (EC 2.1.1.5). It is important to establish its presence or absence in brain, since its substrate, betaine, has recently become established in the treatment of certain diseases involving this organ. 2. It remains unclear whether the reported success of this treatment results from the use of betaine to methylate homocysteine and produce methionine in situ in neural tissue, o r whether the effect is secondary to these same reductions happening in other organs, such as the liver. The former would require the presence of betainehomocysteine methyltransferase in neural tissue. 3. This study demonstrates the complete absence of any activity for this enzyme in the brain of the three species examined. The enzyme was found to be present in both the liver and kidney of man and pig, but only in the liver of the rat. 4. The only source of betaine in cells is via the oxidation of choline. Since the enzymes involved in this conversion have never been shown to exist anywhere other than the mitochondria, it has been assumed that the methyltransferase is also mitochondrial. In this study, it is demonstrated that the enzyme exists only in the cytoplasm of rat liver cells.
Key words: betaine-homocysteine methyltransferase, methionine, neural tissue, nitrous oxide. INTRODUCTION There are two mammalian enzymes capable of remethylating homocysteine, betaine-homocysteine methyltransferase (EC 2.1.1.5) and methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) (Fig. l), the latter, and not the former, Correspondence: Dr J. M. Scott, Department of Biochemistry, Trinity College, Dublin 2, Republic of Ireland.
MITOCHONDRIA
CMOSOL
Protein
Glycine 14
Sarcosine
+
'
11T Dimethylglycine C
gT
rH4F01ate\ SAM
10
Betaine 1
1 I A PP; T P P,
7
5.10CH H Folate
2'
5-CH3H,Folate
zF ."I[
Acceptors
Methylated acceptors
2 I ~-~omocvsteine]
Betaine aldehyde
t
J.
L-Cystathionine
Ch o I i n e
51?
a-Ketobutyrate L-Cysteine
.I
so:-
Fig. 1. Pathways of methionine metabolism. Abbreviations: Pi , inorganic phosphate; PP,, pyrophosphate; H, folate, tetrahydrofolate; S,lOCH,H, folate, 5,lO-methylenetetrahydrofolate; 5CH,H, folate, 5-methyltetrahydrofolate; SAM, S-adenosylmethionine; SAH, Sadenosylhomocysteine. The numbers represent the following specific enzymes or enzymic sequences: 1, methionine S-methyltransferase (EC 2.1.1.12); 2, representative transmethylation reaction; 3, S-adenosylhomocysteinase (EC 3.3.1.1); 4,cystathionine B-synthase (EC 4.2.1.22); 5, cystathionase (cystathionhe y-lyase EC 4.4.1.l); 6, multiple reactions leading from cysteine to sulphate; 7, 5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase, EC 2.1.1.13); 8, choline oxidase (EC 1.1.3.17); 9, betaine-aldehyde dehydrogenase (EC 1.2.1.8); 10, betaine-homocysteine methyltransferase (EC 2.1.1.5); 11, dimethylglycine dehydrogenase (EC 1S.99.2); 12, sarcosine dehydrogenase (EC 1.5.99.1).
M. P. McKeever et al.
552
requiring the cofactor, methylcobalamin. Betaine supplementation has been shown to be an effective treatment in patients with homocystinuria [l,21 and in diseases such as 5,lO-methylenetetrahydrofolatereductase ( E C 1.7.99.5) deficiency [3, 41. Betaine provides an alternative method for producing methionine, and could also have the effect of metabolizing homocysteine which is known to be toxic to several tissues, particularly neural tissue [S]. The activity of betaine-homocysteine methyltransferase has been shown to be confined to liver in rats [6] and in humans it is also present in the kidney [7].Betaine-homocysteine methyltransferase activity was not measurable in rat or monkey brain [8], but a study by Gaull et al. [9] has found this enzyme to be present in human foetal and mature post-mortem brain. On the other hand, others have reported the activity of betaine-homocysteine methyltransferase as being absent from human brain [3, 10, 111. The clinical improvement observed in neural and mental status with betaine treatment could be due either to direct lowering of neural levels of homocysteine in situ, or else to this happening in an organ such as the liver. The former explanation would require confirmation of the observations by Gaull et al. [9] that the methyltransferase is present in neural tissue. Further, it is possible that, although the enzyme might be low or absent in the normal brain, it might be inducible in certain conditions. In the present study, the activity of betaine-methyltransferase was measured in different organs in man, pig and rat. In addition, induction of this enzyme was examined by elevating plasma and tissue levels of homocysteine by inactivating methionine synthase with N,O. The subcellular location of betaine-homocysteine methyltransferase was also investigated. This enzyme catalyses a reaction that is essential for the catabolism of choline. The enzymes in the choline pathway are mitochondrial[12-15]. No adequate studies have been carried out to locate betaine-homocysteine methyltransferase. A n early study [16] indicated that the enzyme was cytosolic, but it was subsequently assumed to be mitochondria1 [13], since the enzymes following it in the choline degradative pathway are known to be mitochon-
drial (Fig. 1).If this was the case, there would have to be a shuttle system for homocysteine to be transported in and methionine transported out of the mitochondria. Definitive identification of the localization of the enzyme would indicate which shuttle system would be expected to occur.
METHODS Chemicals
[rnethyl-'4C]Choline chloride (specific activity 55 mCi/ mmol) was obtained from Amersham International, Amersham, Bucks, U.K. [ 14CH,]Betaine was synthesized essentially as described by Nelson et al. [17]. Choline oxidase was obtained from Sigma Chemical Co. Ltd, Poole, Dorset, U.K. L-Homocysteine was prepared from L-homocysteine thiolactone as described by Duerre & Miller [18]. All other chemicals and standards used throughout were 'Analar' or equivalent grade. Animals
Female Wistar rats (200 g) were maintained in air or in a gaseous mix of 50% N,0/50% air, and were fed a standard chow diet ad libitum. Weanling Landrace pigs were maintained in air or in an atmosphere of 15% N,O, and were fed a diet containing normal nutrients and methionine. Assay methods
Preparations were assayed for betaine-homocysteine methyltransferase activity essentially as described by Finkelstein & Mudd [19]. The following compounds were incubated for 20 min at 37°C in a volume of 1 ml and in the presence of tissue extracts: 35 mmol/l potassium phosphate buffer, pH 7.4, 6.5 mmol/l L-homocysteine, 0.15 mmol/l dithiothreitol, 6.5 mmol/l ['4CH3]betaine (500 x lo3 c.p.m.). The reaction was stopped by placing the reaction mixture on ice and adding 0.25 ml of ice-cold water. The products, methionine and dimethylglycine,
Table 1. Distribution of betaine-homocysteine methyltransferase activity in pig and rat and in human post-mortem tissue Results are means fSD ( n )for the three species indicated. Each sample was assayed in triplicate. Abbreviation: ND, not detectable. Tissue
Liver Kidney Whole brain Frontal lobe Occipital lobe Cerebellum
Thoracic cord Lumbar cord Cervical cord
Betaine-homocysteine methyltransferase activity (nmol h-' mg-' of protein) Human
Pig
32.1 k 10.8 (10) 14.1 k6.3 (10)
62.7 k 4.8 (3) 19.8 k 3.3 (3) ND (3)
ND (10) ND(10) ND (10) ND (10) ND (10) ND(1O)
Rat
Distribution of betaine-homocysteine methyltransferase Table 2. Effect of incubation with neural and liver tissue on betaine-homocysteine methyltransferase activity in rat and in human post-mortem tissue Results are from a single rat and a single human postmortem sample. Each sample was assayed in triplicate. Abbreviation: ND, not detectable. Incubation mix containing:
Brain (300 pl) Liver (300 pl) Liver+brain(300p1+100pl) Liver+brain(300 p1+200 pl) Liver + brain (300 pI + 300 pl)
553
'"1
P
Betaine-homocysteine methyltransferase activity (nmol/h) Human
Rat
ND 184.8 185.3 179.1 173.5
ND 486.4 475.0 469.6 480.4
were separated from excess betaine on Dowex 1X 5 (chloride form) 100-200 dry mesh and a portion was counted in the scintillation fluid hydrofluor (National Diagnostics).A Packard 1500 Tri-Carb liquid scintillation counter was used for measuring radioactivity. T.1.c. was used to identlfy the products of the reaction using plasticcoated silica sheets and a solvent system comprising methanol/6% (w/v) sodium chloride/ammonia (10: 10: 1, by vol.). Preparation of human post-mortem tissue was by the method of Gaull et al. [9]. Liver, kidney, brain and spinal-cord tissue from mature humans with apparently normal organs at post mortem were obtained as soon as possible after death and always within 12 h. Preparation of pig tissue was also by the method of Gaull et al. [9], whereas the method of Finkelstein & Mudd [19] was used for the preparation of rat tissue. Protein was determined by the method of Lowry et al. [20]. The technique of de Duve et al. [21] was used to investigate the subcellular distribution of betaine-homocysteine methyltransferase in rat liver. Marker enzyme activities for subcellular components were measured. The activity of lactate dehydrogenase (EC 1.1.1.27), a marker enzyme for the cytosol, was measured by the method of Bergmeyer & Bernt [22]. Glutamate dehydrogenase (EC 1.4.1.2) and adenylate kinase (EC 2.7.4.3), marker enzymes for the mitochondria1 matrix and inter-membrane space respectively, were measured by the methods of McCarthy et al. [23] and Sottocasa et al. [24]. The method of Heymann et al. [25] was used to measure the activity of glucose-6-phosphatase(EC 3.1.3.9), a marker enzyme for the endoplasmic reticulum, and aryl sulphatase (EC 3.1.6.1), a lysosomal marker, was measured by the method of OFagain et al. [26]. Protein was determined by the method of Markwell et al. [27].
0
20
40
60
80
Time exposed to N,O (h)
Fig. 2. Effect of N,O on betaine-homocysteine methyltransferase (BHMT) activity in rat liver (O),kidney (0) and brain ( A ) . The activity of BHMT was determined in the kidney, liver and brain of rats which had been exposed to N,O over various periods of time. Results are expressed as means If:SD of three separate animals.
activity was detected in any of the three areas of the brain or of the spinal cord that were examined (Table 1). Betaine-homocysteine methyltransferase activity was present in the rat liver, but was not detected in the brain or kidney. In the pig, enzyme activity was found in the kidney and the liver, but again no activity was detected in the brain (Table 1).Incubation of neural tissue with liver tissue indicated that no inhibitor was present in the brain (Table 2). There was a steady increase in the activity of liver betaine-homocysteine methyltransferase in rats exposed to N,O from time 0 to 72 h, but no activity was detected in the brain or kidney (Fig. 2). N,O also increased the activity of betaine-homocysteine methyltransferase in pig liver and kidney, but again no activity was detected in pig brain (Fig. 3). Subcellular distribution of betaine-homocysteine methyltransferase in rat liver
RESULTS
The distribution of betaine-homocysteine methyltransferase essentially followed that of lactate dehydrogenase (Fig. 4). Activity was enriched only in the supernatant fraction, indicating a cytosolic location for the enzyme. The distribution of marker enzymes was consistent with that reported by de Duve et al. [20].
Distribution of betaine-homocysteine methyltransferase in human, rat and pig tissue
DISCUSSION
Activity of this enzyme was only detectable in liver and kidney preparations of the human post-mortem tissue. No
The subcellular distribution of betaine-homocysteine methyltransferase essentially followed that of lactate
M. P. McKeever et al.
554
LDH
BHMT
Adenylate kinase
-
,1 0
20
40
60
80
Aryl sulphatase
G-6-Pase
100
Time exposed to N,O (h) Fig. 3. Effect of N,O on betaine-homocysteine methyltransferase (BHMT) activity in pig liver (O),kidney (0) and brain ( A ) . The activity of BHMT was determined in the liver, kidney and brain of pigs which had been exposed to N,O over various periods of time. Results are expressed as m e a n s k s ~of a single animal assayed on three separate occasions.
N
M L P
M L P
S
1
I
0
N
S
50
100
0
50
I00
of recovered protein
dehydrogenase, a marker enzyme for the cytosolic fraction, indicating a cytosolic location for the enzyme (Fig. 4). The presence of betaine-homocysteine methyltransferase in the cytosol would present a more effective method of removing excess homocysteine, as homocysteine would not have to be transported into, and methionine transported out of, the mitochondria. Betaine promises to be the treatment of choice of homocystinuria [l, 21 and 5,lO-methylenetetrahydrofolate reductase deficiency [3, 41. By reducing the homocysteine concentration while increasing that of methionine, it thereby increases the methionine/homocysteine ratio. The effectiveness of betaine may be due in part to the fact that the enzyme is cytosolic and therefore quickly available when betaine is administered to methylate homocysteine and produce methionine. The distribution of betaine-homocysteine methyltransferase in human, rat and pig tissue indicates that the enzyme is not present in neural tissue. This contrasts sharply with the study of GauU et al. [9], in which the enzyme was reported to be present in adult and foetal post-mortem brain. Since we used the same preparation and assay methods as Gaul1 et af. [9], the reason for this difference is unclear. However, it has been our consistent finding that in the ten human brains studied, the enzyme is absent from this organ (Table 1).Activity was found to be present in rat liver, but not in the kidney or brain (Table 1). Activity was found to be present in the liver and
Fig. 4. Subcellular distribution of betaine-homocysteine methyltransferase (BHMT ) activity in rat liver. A rat liver homogenate was fractionated by differential centrifugation into a nuclear (N), a heavy mitochondrial (M), a light mitochondrial (L), a microsomal (P)and a soluble ( S )fraction, and marker enzymes were measured in each fraction: lactate dehydrogenase (LDH), cytosol; glutamate dehydrogenase (GDH), mitochondrial matrix; adenylate kinase, mitochondrial inter-membrane space; glucose-6phosphatase (G-6-Pase), endoplasmic reticulum; aryl sulphatase, lysosome. Results are expressed as means k SD from three separate preparations and are presented as histograms, where the relative specific activity was plotted against the percentage of recovered protein in that fraction. The relative specific activity of a given component in any fraction was taken as its specific activity or activity/mg of protein divided by the specific activity in whole tissue.
kidney in the human and pig (Table l), but no activity was detected in the brain 'of either species. In order to carry out a direct comparison with other published results [9, 191 different preparation methods were used for the rat and the human. Incubation of neural tissue with liver tissue indicated that no inhibitor was present in the brain (Table 2). The rise in activity of betaine-homocysteine methyltransferase in rat liver exposed to N,O is consistent
Distribution of betaine-homocysteine methyltransferase with that reported by Chanarin et al. [l13 (Fig. 2). There was also a rise in activity in pig liver and kidney (Fig. 3). It is unclear why there is an early induction of betaine-homocysteine methyltransferase activity by N,O in the pig, followed by a fall in activity, but N 2 0 is known to affect several enzymes in folate metabolism [ll].No activity could be detected in the pig brain o r in rat brain and kidney after N,O exposure. Betaine-homocysteine methyltransferase activity is known to increase when methionine synthase is inactivated by N 2 0 , yet no activity could be detected in the brain. This suggested that betaine-homocysteine methyltransferase is neither present normally nor is inducible in this organ. These results indicate that the enzymic capability for remethylation of homocysteine to methionine in brain is quite different from that in liver and kidney. The absence from the brain of this additional pathway for the remethylation of homocysteine contributes to the vulnerability of the nervous system to damage in disorders such as 5,lOmethylenetetrahydrofolate reductase deficiency and homocystinuria. This raises the question as to the mode of action of betaine in the treatment of these two disorders [l, 31. Since betaine cannot be demethylated in neural tissue in the absence of the methyltransferase, it must act by reducing the plasma level of homocysteine after its removal by the liver or kidney. This reduced plasma level would then facilitate clearance of homocysteine from the brain. The absence of betaine-homocysteine methyltransferase in brain tissue also explains why, in pigs maintained in N20, the sole clinical lesion induced is a neuropathy, which we suggest is induced by hypomethylation [28], and why sub-acute combined degeneration occurs in man when methionine synthase is inhibited by either vitamin B,, deficiency [29] o r N,O inhalation [30,31].
REFERENCES 1. Wilcken, D.E., Wilcken, B., Dudman, N.P. & Tyrrell, P.A. Homocystinuria: the effects of betaine in the treatment of patients not responsive to pyridoxine. N. Engl. J. Med. 1983; 309,448-53. 2. Smolin, L.A., Benevenga, N.J. & Berlow, S. The use of betaine for the treatment of homocystinuria. J. Pediatr. 1981; 99,467-72. 3. Erbe, R.W. Inborn errors of folate metabolism. In: Blakely, R.L. & Whitehead, V.M., eds. Folate and pterins. New York Wiley-Interscience, 1986; 3,413-65. 4. Holme, E., Kjellman, B. & Range, E. Betaine for treatment of homocystinuria caused by methylenetetrahydrofolate reductase deficiency. Arch. Dis. Child. 1989; 64, 1061-4. 5 . Mudd, S.H. & Levy, H.L. Disorders of transsulfuration. In: Stanbury, J.B., Wyngaarden, J.B. & Fredrickson, D.S., eds. The metabolic basis of inherited disease. New York: McGraw-Hill Book Company Inc., 1978: 458-503. 6. Finkelstein, J.D., Kyle, W.E. & Harris, B.J. Methionine metabolism in mammals: regulation of homocysteine methyltransferase in rat tissue. Arch. Biochem. Biophys. 1971; 146,84-92. 7. Mudd, S.H., Levy, H.L. & Abeles, R.H. A derangement in B ,2 metabolism leading to homocystinuria, cystathioninemia and methylmalonic aciduria. Biochem. Biophys. Res. Commun. 1969; 35,121-6. 8. Sturman, J.A., Gaull, G.E. & Niemann, W.H. Activities of
555
some enzymes involved in homocysteine methylation in brain, liver and kidney of the developing rhesus monkey. J. Neurochem. 1976; 27,425-31. 9. Gaull, G.E., von Berg, W., Raiha, N.C.R. & Sturman, J.A. Development of methyltransferase activities of human fetal tissues. Pediatr. Res. 1973; 7,527-33. 10. Davies, S.E.C., Chalmers, R.A., Randall, E.W. & Iles, R.A. Betaine metabolism in human neonates and developing rats. Clin. Chim. Acta 1988; 178,241-50. 11. Chanarin, I., Deacon, R., Lumb, M., Muir, M. & Perry, J. Cobalamin-folate interrelations: a critical review. Blood 1985; 66,479-89. 12. Williams, J.N., Jr. Intracellular distribution of choline oxidase. J. Biol. Chem. 1952; 194,139-42. 13. Cook, R.J. & Wagner, C. Glycine N-methyltransferase is a folate binding protein of rat liver cytosol. Proc. Natl. Acad. Sci. U.S.A. 1984; 81,3631-4. 14. Wittmer, A.J. & Wagner, C. Identification of the folatebinding proteins of rat liver mitochondria as dimethylglycine dehydrogenase and sarcosine dehydrogenase. J. Biol. Chem. 1981; 256,4102-8. 15. Wittmer, A J . & Wagner, C. Identification of the folatebinding proteins of rat liver mitochondria as dimethylglycine dehydrogenase and sarcosine dehydrogenase: flavoprotein proteins. J. Biol. Chem. 1981; 256,4109-15. 16. Klee, W.A., Richards, H.H. & Cantoni, G.L. The synthesis of methionine by enzymic transmethylation: VII. Existence of two separate homocysteine methylpherases on mammalian liver. Biochem. Biophys. Acta 1961; 54, 157-64. 17. Nelson, L.D., Brown, N.D. & Wisemann, W.P. Simultaneous assay of choline kinase and choline oxidase in tissue by high-performance cation-exchange chromatography and continuous radioactive detection. J. Chromatogr. 1985; 324,203-8. 18. Duerre, J.A. & Miller, C.H. Preparation of L-homocysteine from L-homocysteine thiolactone. Anal. Biochem. 1966; 17, 310-15. 19. Finkelstein, J.D. & Mudd, S.H. Trans-sulfuration in mammals: the methionine-sparing effect of cystine. J. Biol. Chem. 1967; 242,873-80. 20. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Fohn-phenol reagent. J. Biol. Chem. 1951; 193,265-75. 21. de Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. & Appelmans, F. Tissue fractionation studies: intracellular distribution patterns of enzymes in rat-liver tissue. Biochem. J. 1955; 60,k04-17. 22. Beremever. H.U. & Bernt. E. Lactate dehydrogenase: UVass,; wjth‘pyruvate and NADH. In: Bergmeyer, H.V., ed. Methods of Enzymatic Analysis. London: Academic Press, 1974 VOI. 2,574-9. 23. McCarthy, A.D., Walker, J.M. & Tipton, K.F. Purification of glutamate dehydrogenase from ox brain and liver: evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage. Biochem. J. 1980; 191,605-11. 24. Sottocasa, G.L., Kuylenstierna, B., Ernster, L. & Bergstrand, A. Separation and some enzymatic properties of the inner and outer membranes of rat liver mitochondria. In: Eskabcook, R.W. & Pullman, M.E., eds. Methods in Enzymology. London: Academic Press, 1967: vol. 10, 448-63. 25. Heymann, D., Reddington, M. & Kreutzberg, G.W. Subcellular localisation of 5’-nucleotidase in rat brain. J. Neurochem. 1984; 43,971-7. 26. OFagain, C., Butler, B.M. & Mantle, TJ. The effect of pH on the kinetics of arylsulphatases A and B. Biochem. J. 1983; 213,603-7. 27. Markwell, M., Haas, S.M., Bieber, L. & Tolbert, N. A modification of the Lowry procedure to simpify protein determination in membrane and lipoprotein samples. Anal. Biochem. 1978; 87,206-10.
556
M. P. McKeever et al.
28. Weir, D.G., Keating, S., Molloy, A. et al. Methylation deficiency causes vitamin B,,-associated neuropathy in the pig. J. Neurochem. 1988; 51, 1949-52. 29. Russell, J.S.R., Batten, F.E. & Collier, J. Subacute combined degeneration of the spinal cord. Brain 1900; 23,39-62.
30. Amess, J.A.L., Burman, J.F., Rees, G.M., Nancekievill, D.J. & Mollin, D.L. Megaloblastic haemopoiesis in patients receiving nitrous oxide. Lancet 1978; ii, 339-42. 31. Layzer, R.B., Fishman, R.A. & Schafer, J.A. Neuropathy following abuse of nitrous oxide. Neurology 1978; 28,485.