Flux of the L-Serine Metabolism in Rabbit, Human, and
Dog Livers
SUBSTANTIAL CONTRIBUTIONS OF BOTH MITOCHONDRIAL AND PEROXISOMAL
SERINE:PYRUVATE/ALANINE:GLYOXYLATE AMINOTRANSFERASE*
Hai-Hui
Xue
§,
Takanori
Sakaguchi¶,
Michio
Fujie
,
Hirofumi
Ogawa**, and
Arata
Ichiyama

From the
First Department of Biochemistry, ¶ Second
Department of Surgery, and
Equipment Center, Hamamatsu
University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan and
the ** Department of Biochemistry, Toyama Medical and Pharmaceutical
University Faculty of Medicine, Toyama 930-0194, Japan
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ABSTRACT |
L-Serine metabolism in rabbit,
dog, and human livers was investigated, focusing on the relative
contributions of the three pathways, one initiated by serine
dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine
hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme
system (GCS). Under quasi-physiological in vitro conditions
(1 mM L-serine and 0.25 mM
pyruvate), flux through serine dehydratase accounted for only traces,
and that through SPT/AGT substantially contributed no matter whether
the enzyme was located in peroxisomes (rabbit and human) or largely in
mitochondria (dog). As for flux through serine hydroxymethyltransferase
and GCS, the conversion of serine to glycine occurred fairly rapidly,
followed by GCS-mediated slow decarboxylation of the accumulated
glycine. The flux through GCS was relatively high in the dog and low in
the rabbit, and only in the dog was it comparable with that through
SPT/AGT. An in vivo experiment with
L-[3-3H,14C]serine as the
substrate indicated that in rabbit liver, gluconeogenesis from
L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been
assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.
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INTRODUCTION |
Among the three major enzymes involved in the metabolism of
L-serine in mammalian liver, L-serine
dehydratase (SDH),1
serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and
serine hydroxymethyltransferase (SHMT), the former two are thought to
participate in gluconeogenesis from L-serine (1). Notable
features of SPT/AGT are that this enzyme is located entirely in
peroxisomes in herbivores and humans, largely in mitochondria in
carnivores (2-4), and in both organelles in rodents such as rat, and
in the rat only the mitochondrial enzyme is induced by glucagon (5). It
has been generally accepted from the known overproduction of oxalate in
primary hyperoxaluria type 1, an inborn error of glyoxylate metabolism
caused by a functional deficiency of peroxisomal SPT/AGT (6), that the
enzyme in this organelle plays an important role in the conversion of
glyoxylate to glycine, but the role of mitochondrial and peroxisomal
SPT/AGT in the serine metabolism has not been elucidated. Cytosolic and
mitochondrial isozymes of SHMT (cSHMT and mSHMT, respectively) have
been shown to catalyze the interconversion between serine and glycine
in conjugation with mitochondrial glycine cleavage enzyme system (GCS).
This interconversion occurs especially when there is need for
C1-substituted tetrahydrofolate cofactors or when either
one of these amino acids are used or supplied (1). In the preceding paper (7), we considered SDH, SPT/AGT, and GCS to be the metabolic exits of the serine-glycine pool and showed that SDH is the major enzyme in the metabolism of L-serine in rat liver. The flux
through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution was about
of that through
SDH both in vitro and in vivo. The flux through GCS was comparable with that through SPT/AGT in glucagon-treated rats
(7). However, this pattern of L-serine metabolism in rat liver may not be extrapolated to other animals, because the SDH activity is known to decrease drastically as the body size of animals
increases (8). Although the results obtained with glucagon-treated rats
suggested that mitochondrial SPT/AGT is involved in the metabolism of
L-serine, whether its contribution can be substantial and
whether the enzyme in peroxisomes plays dual roles in the metabolism of glyoxylate and serine remain obscure. This paper deals with the L-serine metabolism in rabbit, dog, and human livers. Dog
and rabbit were chosen as representatives of carnivores and herbivores, respectively.
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EXPERIMENTAL PROCEDURES |
Materials--
Sources of all reagents and male Wistar rats are
described in the preceding paper (7). Japanese white male rabbits
weighing ~2 kg were obtained from a local dealer. Small pieces of
human livers were obtained at the time of surgical operation (hepatic left lobectomy) on three patients who suffered from biliary tract carcinoma or liver metastasis of a sarcoma. Use of the liver samples from human subjects in this study was permitted by the Ethical Committee of Hamamatsu University School of Medicine, and the patients
consented to use of the resected specimens. Small pieces of human liver
specimens were also obtained on autopsy ~5 h postmortem. Small pieces
of dog livers were provided when a research group headed by Dr. Kazuya
Suzuki (First Department of Surgery, Hamamatsu University School of
Medicine) performed an animal experiment according to the Guidelines
for Animal Experimentation of Hamamatsu University School of Medicine.
Assay of Enzymes--
The SDH activity was measured by either
the direct spectrophotometric method used in the preceding paper (7) or
the method described by Ishikawa et al. (9) with
modifications. In the latter method, the reaction was carried out at a
physiological pH, 7.5, in the presence of NADH and lactate
dehydrogenase, and the amount of lactate produced was determined. The
serine:pyruvate aminotransferase activity of SPT/AGT (10) and the SHMT
activity (7, 11) were determined as described in the papers cited. For
the assay of mSHMT, mitochondria were sonicated in 0.125 M sucrose, pH 7.4, containing 1.5 mM imidazole and HCl and
0.05 mM EDTA, and after centrifugation at 105,000 × g for 60 min, the supernatant was used.
Procedures for in Vitro Experiments--
Cytoplasmic extracts,
Mit-Ps suspensions, soluble fractions, and reconstituted cytoplasmic
extracts were prepared from rabbit, human, dog, and rat livers as
described (7). To obtain a mitochondrial suspension, a portion of the
cytoplasmic extract (650 × g supernatant) was
centrifuged at 8200 × g for 10 min, and the pellet was
washed once and suspended in the homogenizing sucrose solution (0.25 M sucrose, 3 mM imidazole and HCl, and 0.1 mM EDTA, pH 7.4) to give a 2.5 ml suspension/g of original liver.
The reactions for the L-serine metabolism and the
decarboxylation from [1-14C]glycine in vitro
were carried out under the same conditions as those described in the
preceding paper (7). Glycine, hydroxypyruvate, pyruvate, and lactate
formed from nonradioactive L-serine and 14CO2 formed from
L-[1-14C]serine or
[1-14C]glycine were determined as described (7).
Procedures for the Infusion Experiment with Rabbits--
Doubly
labeled substrates,
L-[3-3H,14C]serine
(3H/14C ratio, 9.75),
L-[3-3H,14C]lactate
(3H/14C ratio, 6.93), and
D-[3-3H,14C]glycerate
(3H/14C ratio, 8.73), were prepared in the
previous study (7). Rabbits were starved for 48 h before use, and
then the abdominal cavity was opened, and the vena cava was exposed
under Nembutal anesthesia (40 mg/kg of body weight). Each of 0.1 mM L-[3-3H,14C]serine
(3H, 11.41 µCi/ml; 14C, 1.17 µCi/ml), 0.1 mM
L-[3-3H,14C]lactate
(3H, 8.25 µCi/ml; 14C, 1.19 µCi/ml), or 0.1 mM
D-[3-3H,14C]glycerate
(3H, 9.95 µCi/ml; 14C, 1.14 µCi/ml) was
infused into the portal veins of separate rabbits continuously for 15 min at a rate of ~4 ml/15 min, and after termination of the infusion,
the rabbits were allowed to metabolize the infused substrates for
another 5 min as described for the infusion into rats. Then the livers
were isolated and immersed in liquid nitrogen immediately. Radioactive
glucose was isolated from the frozen livers, and its 3H and
14C radioactivities were differentially counted as
described (7).
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RESULTS |
Activities of L-Serine Metabolizing Enzymes in Rabbit,
Human, Dog, and Rat Livers--
In Table
I, the activities of SPT/AGT, SDH, and
SHMT in rabbit, dog, and human livers are compared with those in 24-h
starved and glucagon-treated rat livers. In confirmation of the results of Rowsell et al. (8), the SDH activity in the cytosol in
rabbit liver was 1 order of magnitude lower than that in rat liver, and in dog and human livers it was a barely detectable level. As for SPT/AGT, on the other hand, its serine:pyruvate aminotransferase activity in rabbit liver was approximately twice the glucagon-induced level in the rat, and still higher activities were determined in human
and dog livers. The SHMT activity was largely recovered in the soluble
fraction, 8-15% of the total activity being detected in the
mitochondrial fraction.
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Table I
Activities of L-serine-metabolizing enzymes in rat, rabbit,
dog, and human livers
The SPT activity of SPT/AGT in rat, rabbit, and dog livers was
determined using the cytoplasmic extract (650 × g
supernatant). For determination of the SPT activity in human liver,
small pieces of liver specimens obtained on autopsy ~5 h postmortem
were homogenized, and the 600 × g supernatants from
sonicated homogenates were subjected to the assay. The activities of
SDH and cSHMT were determined with the soluble fraction, and that of
mSHMT was determined using the 105,000 × g supernatant
from the sonicated mitochondrial fraction. The activities are expressed
as µmol/min per g of liver and are averages ± S.D. Values in
parentheses indicate the number of independent determinations.
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The evolution of 14CO2 from
[1-14C]glycine determined as a measure of the GCS
activity (12, 13) was the highest in dog and rat livers, followed by
that in human liver (Fig. 1). In rabbit liver containing the highest cSHMT and mSHMT activities, the activity to decarboxylate [1-14C]glycine was the lowest. These
results altogether suggested that the flux of L-serine
metabolism in rabbit, dog, and human livers is quite different from
that in rat liver described in the preceding paper (7).

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Fig. 1.
Decarboxylation from
[1-14C]glycine in liver mitochondrial
suspensions from 24-h starved (A) and glucagon-treated
(B) rats, rabbits (C), humans
(D), and dogs (E). Mitochondrial
suspensions (8200 × g precipitate) corresponding to 40 mg of starting livers were incubated at 37 °C for 60 min with
various concentrations of [1-14C]glycine (0.05 mCi/mmol),
and the 14CO2 evolved was determined. The
apparent Km for [1-14C]glycine was
determined to be 3-4.5 mM with the mitochondrial
preparations from all the animal species tested. Data obtained with 2.5 mM [1-14C]glycine as the substrate are
presented for comparison between the activities of different animal
species. I, 14CO2 evolved by the
mitochondrial suspensions corresponding to 40 mg of liver. Data are the
mean of two (human) to six (24-h starved rats) separate determinations.
II, in one experiment, the protein content of the
mitochondrial preparations was 1.08, 0.90, 0.66, 0.74, and 0.65 mg/40
mg of starting liver for 24-h starved rat, glucagon-treated rat,
rabbit, human, and dog, respectively. Data are expressed as
14CO2 evolved/mg of mitochondrial protein.
III, in this experiment, the recovery of the glutamate
dehydrogenase activity from cytoplasmic extract to the mitochondrial
fraction was 71.5, 74.9, 95.4, 66.7, and 44.3% for 24-h starved rat,
glucagon-treated rat, rabbit, human, and dog, respectively. Data are
expressed as 14CO2 evolved/40 mg of starting
liver after correction for the recovery of glutamate
dehydrogenase.
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Flux of the L-Serine Metabolism through SPT/AGT in a
Mit-Ps Suspension--
It was not possible to deplete the SDH activity
in rabbit liver soluble fraction with an anti-rat SDH rabbit antibody.
The SDH activity was fairly low in rabbit, human, and dog livers (Table I), but to avoid possible in situ generation of a small
amount of pyruvate from L-serine, we decided to use the
Mit-Ps suspension (25,000 × g precipitate containing
both mitochondria and peroxisomes) rather than the cytoplasmic extract
(650 × g supernatant) for measurement of the flux
through SPT/AGT. SPT/AGT is known to be located largely in mitochondria
in dog liver and exclusively in peroxisomes in human and rabbit livers
(2-4), although the peroxisomal SPT/AGT leaks out to some extent
during the preparation of the Mit-Ps suspension.
To determine the effect of pyruvate concentrations on the flux through
SPT/AGT in a Mit-Ps suspension, the reactions with 5 mM
L-serine and those with 5 mM
L-[1-14C]serine as the substrates were
simultaneously carried out in the absence or presence of various
concentrations of pyruvate, and the flux through SPT/AGT was determined
as the sum of the hydroxypyruvate accumulated and the
pyruvate-dependent increase in the
14CO2 evolution, as in the experiment with rat
liver (7). As shown in Fig. 2, the flux
through SPT/AGT versus the pyruvate concentration curve was
hyperbolic with rabbit and human liver Mit-Ps preparations containing
SPT/AGT in peroxisomes. When dog liver Mit-Ps suspensions containing
SPT/AGT in mitochondria were used, on the other hand, the curve was
sigmoidal. This is probably because pyruvate enters mitochondria
largely by diffusion at its high concentrations, but when the
concentration is low, the entry occurs by a carrier-mediated process,
which appears to be limited by the pyruvate use, as indicated in the
case of rat liver mitochondria (14). As a result, the amount of
hydroxypyruvate formed with dog liver Mit-Ps suspensions in the
presence of quasi-physiological 0.25 mM pyruvate was only
~8% of that in the presence of 2 mM pyruvate and was
less than that formed with human and rabbit liver Mit-Ps preparations
under the same conditions, although dog liver contained a higher
activity of SPT/AGT than human and rabbit livers (compare Table I).

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Fig. 2.
Effect of pyruvate concentration on the flux
of L-serine metabolism through SPT/AGT. Five
mM L-serine and
L-[1-14C]serine were incubated in parallel at
37 °C for 60 min with a Mit-Ps suspension corresponding to 40 mg wet
weight of dog (A), rabbit (B), and human
(C) livers in the absence or presence of the indicated
concentrations of pyruvate. The flux through SPT/AGT was determined
from the amount of hydroxypyruvate accumulated and the
pyruvate-dependent increase in the
14CO2 evolution as described in the text.
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Relative Contributions of the Three Pathways to the Metabolism of
L-Serine in Vitro in Rabbit, Dog, and Human
Livers--
The flux through SPT/AGT was determined with a Mit-Ps
suspension as described above, and that through SDH was evaluated by the amount of pyruvate formed from L-serine with a soluble
fraction as in the case of rat liver (7). For determination of the
amount of L-serine metabolized via glycine, a reconstituted
cytoplasmic extract (a mixture of a Mit-Ps suspension and a soluble
fraction) was incubated with L-[1-14C]serine
in the presence of 0.1 mM tetrahydrofolate and 0.3 mM NADP+ and in the absence of pyruvate, and
the 14CO2 evolved was determined. Pyruvate was
omitted from the reaction mixture based on our previous observation (7)
that the 14CO2 evolution by way of glycine was
independent of the presence of pyruvate, and in this experiment little
pyruvate was accumulated even when L-serine was incubated
with the soluble fraction, as described below. All reactions were
carried out at 37 °C for 60 min with the subcellular preparations
corresponding to 40 mg of liver.
It had been observed in other experiments that the reaction catalyzed
by SHMT in the soluble fractions from rabbit, human, and dog livers
proceeded linearly only for 5-6 min, and the activity was proportional
to the enzyme concentration only when the soluble fractions
corresponding to <10-15 mg of liver were used. Therefore, the amount
of reconstituted cytoplasmic extracts and the incubation time used in
this experiment were out of the linear range with respect to the
cSHMT-catalyzed glycine formation from L-serine. Nevertheless, a large amount of glycine accumulation was observed, irrespective of rabbit, human, or dog liver, after the 60-min incubation (Table II). On the other hand,
the 14CO2 evolution from
L-[1-14C]serine via glycine represented by
the decarboxylation in the absence of pyruvate was very small in rabbit
liver, and even in human and dog livers having higher activities of
GCS, it accounted for only 4-13 and 20-30% of accumulated glycine,
respectively. The in vitro conditions used for serine
metabolism may be out of physiological range with respect to the
glycine metabolism, because glycine accumulated in the reaction mixture
after the 60-min incubation was at most 350 µM, whereas
the glycine concentrations in animal livers have been believed to be on
the order of several µmol/g (15, 16). However, the flux through GCS
measured by the mitochondrial decarboxylation from 2.5 mM
[1-14C]glycine was no more than 3-4-fold of those
determined as the flux through GCS in the metabolism of 1 mM L-serine (compare Fig. 1 and Table II). It
appears that, as in the case of rat liver, cSHMT catalyzes fairly rapid
conversion of serine to glycine, and the rate of the metabolism of
L-serine to CO2 by way of glycine is limited at
the second step involving GCS under the in vitro conditions
used. In fact, the flux through the GCS pathway in the three different
animal species was in accordance with the activity of mitochondria to
decarboxylate [1-14C]glycine, which was the highest in
dog liver, followed by human and rabbit livers (Fig. 1).
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Table II
L-Serine metabolism in vitro in rabbit, human, and dog
livers
Reactions were carried out at 37 °C for 60 min with a Mit-Ps
suspension (for the flux through SPT/AGT) and a reconstituted
cytoplasmic extract (for the flux through SHMT and GCS) corresponding
to 40 mg of liver. Flux through SPT/AGT and that through GCS were
calculated from the amounts of 14CO2 and
hydroxypyruvate formed as described in the text. Glycine was also
determined to show the overall flow of the serine metabolism via
glycine. Data are the representative of two independent experiments
with similar results. Values represent nmol formed or metabolized/60
min, and those in parentheses represent percentages of contributions of
individual pathways. sup, the soluble fraction.
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It is noteworthy that the formation of hydroxypyruvate was evident no
matter whether SPT/AGT is located largely in mitochondria (dog) or
entirely in peroxisomes (human and rabbit). If one assumes that the
physiological concentrations of L-serine and pyruvate in
these animal livers are ~1 and <0.25 mM, respectively,
the relative contributions of the SPT/AGT pathway and that involving SHMT and GCS are calculated to be 96:4 in rabbit liver, 88:12 in human
liver, and 57:43 in dog liver.
Unlike the case of rat liver, pyruvate accumulated by the 60-min
incubation of the soluble fraction from rabbit liver with 0.5-5
mM L-serine was on a barely detectable level or
sometimes too low to be determined with the lactate dehydrogenase
method. In the case of dog and human livers, no accumulation of
pyruvate was detectable, suggesting that the contribution of the SDH
pathway is only traces in the livers of these animals. It is possible that when the SDH activity is very low, pyruvate accumulated during the
60-min incubation of L-serine with the soluble fraction
does not necessarily represent the net pyruvate formation. For example, because the liver contains glutamate and a high activity of soluble alanine aminotransferase, a significant portion of the small amount of
pyruvate formed could be converted to alanine. However, even when the
expected amounts of pyruvate formation under the quasi-physiological conditions (1 mM L-serine) are calculated from
the hepatic SDH activity (Table I) and its Km for
L-serine according to the Michaelis-Menten equation, the
flux through the SDH pathway is assessed to be less than that through
GCS in human and dog livers, and only in rabbit liver could its
calculated contribution be severalfold more than that through GCS.
Relative Contributions of SDH and SPT/AGT to Gluconeogenesis from
L-Serine in Vivo in Rabbit Liver--
To quantify the
relative contributions of the SDH and SPT/AGT pathways to
gluconeogenesis in rabbit liver in vivo,
L-[3-3H,14C]serine,
L-[3-3H,14C]lactate, or
D-[3-3H,14C]glycerate was infused
into the livers of separate rabbits, and then the radioactive glucose
formed was isolated, and its 3H/14C ratio was
determined, according to the principle described in the preceding paper
(7). As in the case of rat liver, 3H was almost lost on
gluconeogenesis from
L-[3-3H,14C]lactate, but it was
largely retained in the glucose formed from D-[3-3H,14C]glycerate (Table
III). Unlike the case of rat liver,
however, the decrease in the 3H/14C ratio was
small on gluconeogenesis from
L-[3-3H,14C]serine, suggesting
that it proceeded mainly via the pathway involving hydroxypyruvate and
D-glycerate. From proportional allotment of the
3H/14C ratio of glucose obtained with
L-[3-3H,14C]serine as the
gluconeogenic substrate between the two control values (those obtained
with L-[3-3H,14C]lactate and
D-[3-3H,14C]glycerate),
the SPT/AGT pathway was estimated to account for ~90%.
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Table III
3H/14C ratio of glucose formed from
L-[3-3H,14C]serine in 48-h starved
rabbits
Experimental details are given under "Experimental Procedures."
L-[3-3H,14C]Serine,
L-[3-3H, 14C]lactate, or
D-[3-3H, 14C]glycerate was infused into
the portal veins of separate rabbits (one each), and in each case the
isolation from the livers of radioactive glucose and determination of
its 3H/14C ratio were carried out in duplicate. Data
presented are the means of the duplicate determinations with similar
results.
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DISCUSSION |
In this study, we have presented in vitro and in
vivo data suggesting that, in rabbit, dog, and human livers,
SPT/AGT substantially contributes to the metabolism of
L-serine, whereas the contribution of SDH is fairly small.
This pattern of L-serine metabolism is in contrast to the
observation that in rat liver the contribution of SDH is the largest,
and the flux through SPT/AGT accounts for only traces unless this
enzyme has been induced by a previous administration of glucagon (7).
Such species-specific patterns of the L-serine metabolism
approximately agree with the observed activities of serine-metabolizing
enzymes in respective animals (Table I). Our results are compatible
with the observation by Rowsell et al. (8) that in rabbit
and dog livers the SPT/AGT activity is more than six times higher, and
the SDH activity is much lower than the respective activities in rat liver.
It is noteworthy that SPT/AGT is involved in the L-serine
metabolism, no matter whether the enzyme is largely located in
mitochondria (dog liver) or entirely in peroxisomes (rabbit and human
livers). It has been expected that mitochondrial SPT/AGT plays a role
in L-serine metabolism, because in the rat, the flux
through SPT/AGT in gluconeogenesis from L-serine was
apparent only when liver mitochondrial SPT/AGT had been induced by
glucagon (7). This was corroborated in the present study by
demonstrating in in vitro experiments under
quasi-physiological conditions that the flux through SPT/AGT accounts
for a large portion of L-serine metabolized in dog liver.
These results are in accordance with the observation of Beliveau and
Freedland (17) that serine is mainly metabolized via transamination in
hepatocytes isolated from the cat, another carnivore. As for the
peroxisomal SPT/AGT, on the other hand, an important role in the
conversion of glyoxylate to glycine has been indicated from the studies
on primary hyperoxaluria type 1 (6). In the present study, the
peroxisomal SPT/AGT was also shown to participate in
L-serine metabolism in vitro (Table II). In
addition, in the case of rabbit liver, an infusion experiment with
L-[3-3H,14C]serine suggested that
the flux through SPT/AGT contributes to gluconeogenesis from
L-serine in vivo and accounts for as much as
~90% of it (Table III). It is evident that peroxisomal SPT/AGT plays
dual roles in the metabolism of serine and glyoxylate. It is possible,
although it has not yet been experimentally proved, that mitochondrial
SPT/AGT also plays a role in the metabolism of glyoxylate.
L-Hydroxyproline has been shown to be metabolized to
glyoxylate in mitochondria (or peroxisomes, 18,000 × g
precipitate; Ref. 18), and glyoxylate thus formed was proposed to be
converted to glycine by mitochondrial SPT/AGT, although the quantities
of the hydroxyproline-derived glyoxylate must be small (1, 19). SPT/AGT
is thus a unique enzyme of dual organelle localization, and it is
possible that the enzyme in either organelle plays dual roles,
gluconeogenesis from serine and conversion of glyoxylate to glycine by
transamination. Such dual functions probably come from the fact that
serine:glyoxylate aminotransferase (EC 2.6.1.45), the plant counterpart
of animal SPT/AGT (EC 2.6.1.44/2.6.1.51), catalyzes transamination
between serine and glyoxylate in the photorespiratory nitrogen cycle
(20), forming hydroxypyruvate and glycine, the latter being converted
back to serine by a mitochondrial enzyme system. It is unlikely that
glyoxylate is supplied in the animal livers as abundantly as in the
photorespiration in plant leaves. Therefore, the hepatic serine
metabolism catalyzed by SPT/AGT may not be necessarily coupled with the
glyoxylate-to-glycine conversion, and more versatile pyruvate may also
be used as an amino acceptor from serine, although the affinity of the
enzyme for glyoxylate is much higher than that for pyruvate, as
demonstrated by much lower Km for glyoxylate (10 µM) than that for pyruvate (480 µM) of the
rat liver enzyme at pH 7.4 (21).
Because plant tissues contain significant amounts of glycolate (22),
and the conversion of glycolate to glyoxylate takes place in liver
peroxisomes, the peroxisomal location of SPT/AGT may be favorable for
herbivores to convert the glycolate-derived glyoxylate to glycine and
to prevent harmful overproduction of oxalate. For carnivores, on the
other hand, there may be less need of the peroxisomal location of
SPT/AGT, because meats contain much less glycolate (22). With respect
to its role in the disposal of serine demonstrated in this study,
SPT/AGT in either of the two organelles appears to function with
similar efficiency. Thus the necessity and advantages to carnivores of
having SPT/AGT in mitochondria are not yet fully understood and need
further studies.
As for the metabolism of L-serine via glycine, the overall
flux observed with reconstituted cytoplasmic extracts from rabbit, human, and dog livers was similar to that in the case of rat liver. As
proposed in the preceding paper (7), the SHMT-catalyzed interconversion
between serine and glycine may contribute, at least in part, to the
maintaining of their intracellular concentration balance, in response
to supply or use of these amino acids and tetrahydrofolate coenzymes,
and the degradation of glycine by GCS may be considered as a metabolic
exit of not only glycine but also L-serine. This view was
further supported in the present study by showing that, irrespective of
the animal species tested, the conversion of serine to glycine
catalyzed by cSHMT occurred fairly rapidly, followed by the
GCS-mediated slow decarboxylation of the accumulated glycine. However,
the flux through GCS as well as that through SDH and SPT/AGT varied
considerably from animal to animal. In dog liver in which the activity
of GCS was relatively high, the contribution of the flux through GCS
was comparable with that through SPT/AGT, but in rabbit and human
livers the GCS pathway accounted for only a small portion of
L-serine metabolized (Table II). It has been previously
noted that the activity of GCS is relatively small, and the apparent
low activity may have some rationale, because glycine is an important
amino acid that is used in many ways in the cells (12). Although the
flux through SPT/AGT is one of the major metabolic exits of serine in
rabbit, human, and dog livers, its activity is also relatively small, consistent with the previous observation by Felig et al.
(23) that serine, as well as glycine, is less effective than alanine as
a precursor for hepatic gluconeogenesis in humans.
Summarizing all the data and discussions presented in this paper
together with those in the preceding one (7), we believe that SDH,
SPT/AGT, cSHMT, mSHMT, and GCS work as schematically shown in
Fig. 3 in the hepatic metabolism of
L-serine and glycine in rats, rabbits, humans, and
dogs.

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Fig. 3.
Proposed role of individual enzymes involved
in the metabolism of L-serine and glycine in mammalian
livers. GCS, SDH, and SPT/AGT are regarded as metabolic exits of
the serine-glycine pool (shaded ellipse), the intracellular
concentration balance of which is maintained by SHMTs and GCS.
Solid arrows show the direction of each reaction in
vivo and do not mean that the reactions are irreversible in
vitro. The metabolic fates of C1 units derived from
L-serine and glycine are cited from Appling (24) and Wagner
(25). OH-Pyr, hydroxypyruvate; Pyr, pyruvate;
THFc and THFm, cytosolic and mitochondrial
tetrahydrofolate, respectively; CH2-THFc and
CH2-THFm, cytosolic and mitochondrial
5,10-methylenetetrahydrofolate, respectively; fMet-tRNA,
formylmethionyl tRNA; dTMP, thymidylate.
|
|
 |
ACKNOWLEDGEMENTS |
We are indebted to Prof. Satoshi Nakamura
(Second Department of Surgery, Hamamatsu University School of Medicine)
and Dr. Kazuya Suzuki (First Department of Surgery, Hamamatsu
University School of Medicine) for their kind supply of human and dog
liver specimens, respectively.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Scholarship student of the Heiwa Nakajima Foundation (1995-1996)
and the Ito Foundation for International Education Exchange (1997-1998).

To whom correspondence should be addressed: First Department of
Biochemistry, Hamamatsu University School of Medicine, 3600 Handa-cho,
Hamamatsu, Shizuoka 431-3192, Japan. Tel.: (81)53-435-2322; Fax:
(81)53-435-2323.
 |
ABBREVIATIONS |
The abbreviations used are:
SDH, L-serine dehydratase (EC 4.2.1.13);
SHMT, serine
hydroxymethyltransferase (EC 2.1.2.1);
mSHMT, mitochondrial isozyme of
serine hydroxymethyltransferase;
cSHMT, cytosolic isozyme of serine
hydroxymethyltransferase;
SPT/AGT, serine:pyruvate/alanine:glyoxylate
aminotransferase (EC 2.6.1.51/EC 2.6.1.44);
GCS, glycine cleavage
enzyme system (EC 1.4.4.2-1.8.1.4-2.1.2.10);
Mit-Ps, subcellular
fraction containing mitochondria and peroxisomes.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.