Clinical Science (1985) 69, 281-292
287
Demonstration of methionine synthetase in intestinal mucosal
cells of the rat
J . N. KEATING, D . G. WEIR
AND
J.
M . SCOTT
Departments of Biochemistry and Clinical Medicine, Trinity College, Dublin, Ireland
(Received 29 October 1984118 March 1985; accepted 2 April 1985)
summary
1. Methionine synthetase was measured in the
mucosal cells of the rat duodenum, jejunum and
ileum by a previously employed method for
mucosal cell isolation. No activity was found in
these cells.
2. When a dual buffer system for the isolation
of villous and crypt cell population was substituted, however, methionine synthetase was found to
be active in the duodenum, jejunum and ileum,
both in the villous and crypt cell populations. The
activity was significantly higher in the crypt cells
than in the villous cells throughout the intestine,
and higher levels were found in the ileum than in
the duodenum or jejunum.
3. As had been previously reported for the rat
liver, nitrous oxide in vivo reduced the enzyme
activity in both the villous and crypt cell populations, suggesting a role in vivo for the enzyme. We
conclude that methionine synthetase is both
present and active in the small intestinal mucosal
cells of the rat.
Key words: cobalamin, methionine synthetase,
mucosal cells, nitrous oxide, vitamin BIZ.
Abbreviation: S-CH3-H4PteGlu, Ns-methyltetrahydrofolate.
Introduction
Methionine synthetase (EC 2.1.1.13) catalyses the
final step in the cobalamin-dependent biosynthesis
of the amino acid, methionine, in mammalian and
bacterial systems [l-31. This step involves the
methyl group transfer from the methyl donor
Correspondence: Dr J. M. Scott, Department of
Biochemistry, Trinity College, University of
Dublin, Dublin 2, Ireland.
N5-methyltetrahydrofolic acid (5-CH3-H&eGlu)
to homocysteine [4]. The reaction requires methylcobalamin and S-adenosylmethionine and is dependent on a reducing system for activity [S].
In the cell, methionine synthetase has two
functions. The first is the biosynthesis and
conservation of methionine. The second is the
demethylation of S-CH3-HJ’teGlu yielding Hr
PteGlu. In the course of the reaction the free
HJ’teGlu returns to the intracellular folate pool
to serve in the further folate-dependent onecarbon transfer reactions. In cobalamin deficiency,
failure to demethylate S-CH3-H&eGlu results in
the prevention of thymidylate and purine biosynthesis, leading to the megaloblastic changes
seen in rapidly proliferating tissue such as bone
marrow.
Such megaloblastic changes have been observed
in the small intestinal mucosal cells in pernicious
anaemia in man [6]. The presence of these changes
suggests two possibilities. These cells could be
affected by compromised methionine synthetase
elsewhere in the body, e.g. via a decreased supply
of reduced folates or methionine coming to the
mucosal cells via the plasma. Alternatively a
hitherto undetected and functionally important
methionine synthetase might exist in intestinal
mucosal cells.
Methionine synthetase activity has been reported in tissue extracts of the liver, kidney and
brain from a variety of animals, including man
[2, 71. Studies in the rat have shown the enzyme
to be present in every cell assayed with the
exception of the small intestinal mucosal cell [2].
It has been demonstrated that preparation of
intestinal cell fractions by a dual buffer system
results in an excellent recovery of intact villousrich and crypt-rich cell populations [8]. This
present investigation was designed to utilize this
technique for a reinvestigation of the methionine
288
J. N . Keating et al.
synthetase levels in the rat intestinal mucosal
cells,
The anaesthetic, nitrous oxide, is a useful
experimental tool for rapidly inducing a cobalamin
inactive state [9]. It does this by oxidizing the
cobalt atom of the transitional metal complex,
thus inhibiting methionine synthetase, leading to a
decreased demethylation of 5-CH3-H$teGlu [ 10121. The methionine synthetase levels in the
intestinal mucosal cells were also investigated after
nitrous oxide inhalation in the rat.
Experimental procedure
Materials
buffer B was decanted. This cell preparation contained enriched villous cells as indicated by the
villous cell marker enzyme, alkaline phosphatase.
The presence of alkaline phosphatase was verified
by a cytochemical staining procedure as described
in Sigma Bulletin 85 (1975). Owing to the nature
of this procedure, results will not be presented
quantitatively.
The segments were flushed with NaCl solution
(0.9%. w/v) to remove residual cells from the
lumen, refilled with buffer B and incubated for a
further 20 min for the isolation of enriched
crypt cells. The cells were pelleted, at 500g,
washed three times in NaCl solution (0.976, W/V)
and prepared for methionine synthetase measurement.
5-['4C]Methyltetrahydrofolic acid, barium salt
(45 mCi/mmol), and ~-[methyl-'~C]methionine
Methionine synthetase assay
(56.7 mCi/mmol) were supplied by Amersham
Methionine synthetase activity was determined
International, U.K. L-Homocysteine thiolactone,
by measurement of the formation of ['4C]methioused in the preparation of L-homocysteine, and all
nine from 5-['4CH,]H$teClu. Liver or mucosal
other chemicals used in these experiments,
cells were homogenized on ice in 9 vol. of
including unlabelled 5-CH3-H,PteGlu and soybean
potassium phosphate (0.01 mol/l) buffer, pH 7.4,
trypsin inhibitor, were obtained from Sigma
followed by centrifugation at 20 000 g for 80 min
Chemical Company, Poole, Dorset, U.K.
at 4°C [ l l ] as modified by Koblin er al. [12].
Aliquots of the supernatant (100 p l j were incuAnimals and treatment
bated in 100 p1 of substrate mixture containing
cyanocobalamin (200 nmol/l), dithiothreitol (58
Male Wistar rats weighing 150-200 g were used.
mmol/l), S-adenosylmethionine (0.5 mmol/l),
Rats in the nitrous oxide study were placed in
homocysteine (15 mmol/l) (prepared daily from
sealed chambers, through which was flowing
its thiolactone derivative), P,P-mercaptoethanol
N,O/O, (50:50, v/v) for 24 h at a flow rate of
(14 mmol/l), 1 mmol/l 5-['4CH3]H$teClu (0.25
0.5 litre/min, during which time they fed as
pCi) and sodium phosphate buffer (1 75 mmol/l),
control rats.
pH 7.5. The reaction mixtures were incubated for
1 h at 37°C. Under these conditions, the rate of
Isolation of intestinal crypt and villous cells
the reaction was linear with time and enzyme
concentration. The reaction was terminated by the
Villous and crypt cells were obtained by the
addition of 0.8 ml of ice-cold water and the
dual buffer technique as described by Merchant &
mixture was passed through an AG-l-x8 anionHeller [8]. Segments of the duodenum, jejunum
exchange resin (Cl- form) obtained from Biorad,
and ileum, 4 cm, 15 cm and 10 cm respectively,
w h c h retains the 5-['4CH3]HJ'teClu. The columns
were excised and rinsed thoroughly in ice-cold
were further washed with 1 ml of water and the
NaCl solution (0.9%, w/v>. Further manipulations
were carried out at room temperature. The gut
[ ''C]methionine formed was measured by counting 1 ml of the pooled effluent in 10 ml of
segments were then closed at one end and
distended with buffer A, containing KC1 (1.5
toluene/Triton-X-l OO (2 : 1, v/v) containing PPO
(2,5-diphenyloxazole) (2.67 g/l) in a Packard
mmol/l), NaCl (96 mmol/l), sodium citrate (25
mmol/l), KH2P04 (8 mmol/l) and Na2HP04 liquid scintillation counter. Enzyme activity was
(5.6 mmol/l), adjusted t o pH 7.3. The open ends
expressed as nmoles of product formed per hour
of the segments were closed, the segments
per mg of protein. The protein concentration of an
immersed in NaCl solution (0.9%, w/v) and incualiquot of the liver cell supernatant (10 plj and the
mucosal cell supernatant (20 p1) was estimated
bated in a shaking water bath at 37OC. After 15
by the Lowry method [13] with a bovine serum
min, buffer A was decanted and the segments
were filled with buffer B containing NaCl (0.14
albumin protein standard in the concentration
range 0-250 ng/ml (w/v).
mmol/l), KCl (2.69 mmol/l), Na2HP04 (8.1
Each enzyme assay was undertaken in duplimmol/l), KH2P04 (1.47 mmol/l), EDTA (1.5
cate; values differing by more than 5% were not
mrnol/l) and dithiothreitol (0.5 mmol/l), adjusted
included
to pH 7.4. After incubation at 37°C for 40 min
Intestinal methionine synthetase
289
TABLE1. Methionine synthetase activity in intestinal mucosal cells of the rat
Mucosal cells were isolated by the dual buffer method, and values are expressed as
means f SEM with the numbers of animals in parentheses.
Activity (nmol h'' mg-' of protein)
P*
(Student's paired t-test)
Duodenum
Jejunum
Ileum
Villous cell
Crypt cell
0.78 i 0 . 0 2 (6)
0.74 i 0.06 (9)
0.90 i 0.05 (9)
1.20 i 0 . 0 8 (6)
1.12i0.04 (6)
1.73 k0.17 (6)t
<0.001
<0.02
<0.001
* Significant differences between villous
t
cell levels and crypt cell level for each region of the small
intestine.
Significance of difference between activities in ileal crypt cell and in duodenal crypt cell (P<
0.01) and in jejunal crypt cell (P< 0.01).
TABLE2. Methionine synthetase activity in the liver and jejunal mucosal cell of the
rat assayed in the presence and absence of mucosal ce2l homogenute and trypsin
inhibitor
Each value represents the mean f SEM derived from the study of four rats. n.d., Not
detectable.
Activity
(nmol h-' mg-' of protein)
I
I1
I11
IV
V
VI
VII
VIII
IX
X
XI
Liver
Mucosal cells isolated by differential scraping method [ 2 ]
Mucosal cells isolated by dual buffer method [ 8 ]
Liver + supernatant of I1 (10 pl)
Liver supernatant of 111 (10 pl)
Supernatant of 111 (100 pl) + supernatant of I1 (10 pl)
Liver supernatant of I1 (10 pl) + antitrypsin
Supernatant of I1 antitrypsin
Supernatant of I1 + antitrypsin added to homogenizing buffer
Liver + buffer A*
Liver buffer A* (10 pl) antitrypsin
+
+
+
+
+
* Buffer A, decanted from gut segment before mucosal
Results
Methionine synthetase activity in mucosal cell
populations
Methionhe
Wnthetase was assayed in the
villous-rich and crypt-rich cell populations in the
duodenum, jejunum and ileum of the rat, obtained
by the dual buffer procedure [s].The results are
summarized in Table 1.
The enzyme was active in both the villous and
crypt cells in each region of the small intestine.
Although the differences in activity between
villous cell populations from the duodenum and
jejunum were not statistically significant, both
were significantly less than those of the ileum.
In addition the enzyme activity in the crypt-rich
cell populations was significantly greater than in
the villous cell populations in the duodenum,
jejunum and ileum, and again the ileal activity in
3.601.0.08
n.d.
1.40.1 0.43
0.66 i 0.09
3 . 7 5 i 0.08
0.06 i 0.39
3.55 i 0.09
n.d.
1.38i 0.41
1.28t 0.26
3.50i 0.09
cell isolation.
the crypt cell population was greater than in both
the duodenal and jejunal populations.
Methionine synthetase activity in small intestinal
mucosal cells isolated b y mucosal scraping
Previous attempts to detect methionhe SynthetaSe in intestinal Cells had involved Cells isolated by
scraping the jejunal mucosa [2]. By this method
we did not find active enzyme in these cells as
had previously been reported [2]. To determine
if this absence of activity was due to the inactivation of the enzyme by proteolytic digestion or
some other type of inhibition an aliquot (1Opl)
of this preparation was incubated with a liver
enzyme preparation and assay buffer. The activity
of the enzyme was dramatically reduced from
3.60 0.08 nmol h-' mg-' of protein to 0.66 ?r
0.09 nmol h-' mg-' of protein (Table 2). When our
*
J. N. Keating et al.
290
preparation obtained by the dual buffer technique
was incubated with the liver preparation, however,
no reduction in activity ensued. A soybean extract
trypsin inhibitor [l mg/rnl (w/v) of assay buffer]
prevented the inactivation of the liver enzyme by
the mucosal cell preparation obtained by the
mucosal scraping procedure. In a similar manner
the activity of mucosal cell preparation, obtained
by the dual buffer technique, could be inactivated
by the mucosal scraped enzyme preparation and
once again this inactivation could be prevented by
the addition of the trypsin inhibitor to the assay
mix.
In order to demonstrate that the trypsin-like
inactivator of methionine synthetase was removed
in the dual buffer mucosal cell isolation procedure,
an aliquot (10 pl) of the buffer A, with which the
gut segment had been initially distended before
villous and crypt cell isolation, was added to the
liver enzyme assay mix. Once again methionine
synthetase was inactivated and furthermore the
inactivation was prevented by the addition of the
trypsin inhibitor.
The mucosal cell preparation, obtained by
mucosal cell scraping, was assayed when trypsin
inhibitor was added either before homogenization
(1 mg/ml in homogenizing buffer) or in the assay
mix as described. The enzyme was significantly
more active in the former (1.38 f 0.41) than in the
latter (0.19 0.10) instance (Table 2).
Methionine synthetase activity in monkey, pig,
rabbit and guinea-pig mucosal cells
The methionine synthetase activity in the
mucosal, villous and crypt cells was investigated
in the duodenum, jejunum and ileum of the
monkey and pig and in the jejunum of the rabbit
and guinea pig. In each instance mucosal cells
were isolated by the dual buffer technique. The
enzyme was active in each mucosal cell type of
each species studied.
Discussion
Methionine synthetase, hitherto reported as being
absent from the gut, was found to be active in
each section of the small intestine, both in the
villous and crypt cell populations. This mucosal
cell enzyme was inactivated by nitrous oxide and
was also found in jejunal villi and crypts of the
monkey, pig, rabbit and guinea pig in addition to
the rat.
In this study, as had been previously demonstrated [2], methionine synthetase was not detected in intestinal cells isolated by scraping the
jejunal mucosa. In addition, with liver enzyme, the
presence of an inactivator of methionine synthetase
was confirmed in this mucosal cell extract, this
inactivation being prevented by the addition of a
trypsin inhibitor.
With a dual buffer technique, as previously
described [8], to isolate intact mucosal villous and
Effect of nitrous oxide on methionine synthetase
crypt cells, methionine synthetase activity was
activity in the mucosal cells
measured in these cells. No methionine synthetase
Rats were maintained in nitrous oxide for 24 h, inactivation was observed when the intestinal
after which time the levels of methionine synthe- mucosal cell extract, isolated by the dual buffer
tase in the liver and the jejunal mucosal cell method, was co-incubated with the liver enzyme
extract. The inactivating agent appeared to be lost
preparations were investigated. The results are
in the mucosal cell isolation procedure.
summarized in Table 3. The activity of the enzyme
When the buffer A, which was decanted from
was decreased by approximately 70-80% in the
villous and crypt cells of the jejunum, comparable the gut segments before mucosal cell isolation, was
to a decrease by 80% in the livers of such NzO- co-incubated with the liver enzyme it proved to
contain an inactivating agent, which had been
treated rats.
*
TABLE 3 . Effect o f nitrous oxide on methionine synthetase activity in the liver and
jejunal mucosal cells of the rat
Mucosal cells were isolated by the dual buffer method [8] and values are expressed as
means k SEM with the numbers of animals in parentheses.
Activity (nmol h-' rng-' of protein)
P*
(Student's paired r-test)
Liver cell
Jejunal villous cell
Jejunal crypt cell
Air
N*O
4.20 t0.23 (6)
0.74 i 0.56 (9)
1.12+0.04 (6)
0.83 20.05 (6)
0.25 20.09 (6)
0.26 t0.07 (4)
* Significance of difference between airexposed and N,O-exposed groups.
<0.001
<0.001
<0.001
Intestinal methionine synthetase
29 1
TABLE4. Methionine synthetase activity in the intestinal mucosal cells of the monkey,
pig, rabbit and guinea pig
Dashes indicate that the enzyme activity was not assayed. n = number of animals.
~~~
~
~~
Activity (nmol h-' mg-' of protein)
Monkey
(n = 1)
Pig
( n = 2)
Rabbit
(n = 2)
Villous cell
Duodenum
Jejunum
Ileum
0.77
0.36
0.52
0.72
0.50
0.18
-
-
0.71
0.88
Crypt cell
Duodenum
Jejunum
Ileum
0.39
0.26
0.76
0.46
0.53
0.33
-
-
0.82
1.17
present in the gut lumen and removed from the
cell preparation by the 15 min incubation and
subsequent decanting of the supernatant. This
inactivating agent activity was prevented by a
trypsin inhibitor. Furthermore, the enzyme could
be measured in the mucosal scraped preparation
when a trypsin inhibitor was added before
homogenization (Table 2), suggesting that inactivation of methionine synthetase due to trypsin had
occurred during the homogenization and centrifugation steps of the enzyme preparation and
before the enzyme assay stage.
The dual buffer method of mucosal cell isolation led to the measurement of methionine synthetase in the duodenum, jejunum and ileum both in
the villous-rich and crypt-rich cell populations.
The enzyme appeared to be mors active in the
crypt-rich cell population. One might interpret this
as being a result of an increased requirement in the
more rapidly dividing crypt cell for the provision
of demethylated folates for its more active purine
and thymidylate biosynthesis, associated with cell
division.
The ileal mucosal cell enzyme was more active,
both villous and crypt, than the duodenal or
jejunal mucosal cell enzyme. This may be a
function of its role in cobalamin absorption. The
cobalamins are absorbed .in the ileum in man
[14] and the middle and distal gut segments have
been implicated in the rat [ 151.
Nitrous oxide has been shown to inactivate rat
liver methionine synthetase [11 , 121. In this study,
the intestinal mucosal cell enzyme was also inactivated by nitrous oxide, suggesting that the cobalamin factor, as in the case of the liver enzyme, is
active when its coenzyme, cobalamin, in its
reduced state, cob(1)alamin. Because the food
intake over 24 h was comparable between the rats
-
-
Guinea pig
(n = 2)
-
-
maintained in air and NzO, it is unlikely that the
decline in enzyme activity in the gut of the NzOtreated animals was a function of diet. Inactivation
by N20 depends on the enzyme being used for
catalysis during the period of NzO exposure. This
decrease in activity subsequent to such exposure
suggests a role in uiuo for the mucosal cell enzyme.
A detailed account of changes in the jejunal
mucosa in pernicious anaemia has been reported
[6]. If such jejunal mucosal cells did not contain
the
methylcobalamin-dependent methionine
synthetase it would be difficult to explain how
such cells could be affected by compromised
methylcobalamin levels, leading to the restricted
folate-dependent purine and thymidylate biosynthesis. It has now been shown that one can
measure this important folate-dependent enzyme
in the gut mucosal cell, not only in the rat, but in
the monkey, pig, rabbit and guinea pig.
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