Báo cáo Y học: Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase docx

Ontogeny and subcellular localization of rat liver mitochondrial
branched chain amino-acid aminotransferase
Nimbe Torres
1
, Carolina Vargas
1
, Rogelio Herna
´
ndez-Pando
2
,He
´
ctor Orozco
2
, Susan M. Hutson
3
and
Armando R. Tovar
1
1
Departamento de Fisiologı
´
a de la Nutricio
´
n, Instituto Nacional de Ciencias Me
´
dicas y Nutricio
´
n ‘Salvador Zubira
´
n’, Me

´
xico;
2
Departamento de Patologı
´
a Experimental, Instituto Nacional de Ciencias Me
´
dicas y Nutricio
´
n ‘Salvador Zubira
´
n’,Me
´
xico;
3
Department of
Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina, USA
Branched chain amino-acid aminotransferase (BCAT)
activity is present in fetal liver but the developmental
pattern of mitochondrial BCAT (BCATm) expression in rat
liver has not been studied. The aim of this study was to
determine the activity, protein and mRNA concentration of
BCATm in fetal and postnatal rat liver, and to localize this
enzyme at the cellular and subcellular levels at both
developmental stages. Maximal BCAT activity and BCATm
mRNA expression occurred at 17 days’ gestation in fetal rat
liver and then declined significantly immediately after birth.
This pattern was observed only in liver; rat heart showed a
different developmental pattern. Fetal liver showed intense
immunostaining to BCATm in the nuclei and mitochondria

of hepatic cells and blood cell precursors; in contrast, adult
liver showed mild immunoreactivity located only in the
mitochondria of hepatocytes. BCAT activity in isolated fetal
liver nuclei was 0.64 mU
:
mg
21
protein whereas it was
undetectable in adult liver nuclei. By Western blot analysis
the BCATm antibody recognized a 41-kDa protein in fetal
liver nuclei, and proteins of 41 and 43 kDa in fetal liver
supernatant. In adult rat liver supernatant, the BCATm
antibody recognized only a 43-kDa protein; however,
neither protein was detected in adult rat liver nuclei. The
appearance of the 41-kDa protein was associated with the
presence of the highly active form of BCATm. These results
suggest the existence of active and inactive forms of BCAT
in rat liver.
Keywords: branched-chain amino acids; mitochondria;
nuclei; ontogeny.
The branched-chain amino acids (BCAA) leucine, iso-
leucine, and valine are required mainly for body protein
synthesis. The initial enzymes for catabolism of the BCAA
are regulated differently from other amino-acid degrading
enzymes. The first step in the degradation of these amino
acids is a reversible transamination catalyzed by the
branched chain amino-acid aminotransferase (BCAT;
EC 2.6.1.42). The products of this reaction are the
corresponding branched chain 2-oxo acids that can be
reaminated to their corresponding amino acids [1], or

irreversibly decarboxylated by the branched-chain 2-oxo
acid dehydrogenase complex (BCODC) forming the
corresponding acyl CoA derivates. In mammals there are
two BCAT isoenzymes, a mitochondrial (BCATm), and a
cytosolic (BCATc) form [2,3]. In the rat BCATm is the
predominant isoenzyme, and it is found in almost all tissues
with the highest activity in pancreas and stomach,
intermediate activity in heart and kidney, low activity in
skeletal muscle and skin and negligible activity in adult
liver. BCAT activity is accompanied by a similar pattern of
BCATm mRNA expression [4]. The cytosolic form is
restricted to brain, ovary and placenta [5]. BCATm cDNA
encodes a polypeptide of 366 amino acids preceded by a pre-
sequence of 27 amino acids with a molecular mass of the
mature protein of 41.2 kDa. The mature rat sequence is 82%
and 95% identical to the human and murine BCATm
respectively [6] and 82% identical to sheep BCATm [7]. In
contrast with other hepatic amino-acid degrading enzymes
[4], BCATm expression is not regulated by glucagon,
glucocorticoids or high dietary protein. However, BCATm
mRNA expression is highly induced in lactating mammary
tissue and declines rapidly after weaning [8,9].
Previous studies have shown that fetal rat liver, in contrast
with adult rat liver, has BCAT activity but that this declines
rapidly after birth [10,11]. This decrease was associated
mainly with a decrease in the volume fraction of
hematopoietic cells in fetal rat liver [11] assuming that the
enzyme activity was confined to only hematopoietic cells
and not to hepatic cells. However, studies with freshly
isolated hepatocytes from 18-days gestation fetal rats and

fetal hepatocytes cultured for 2 days showed BCAT activity,
indicating the possibility that not only the hematopoietic
cells were responsible for BCAT activity but that fetal
hepatocytes may contribute also to the enzyme activity [12].
In the present study, we measured the BCAT activity,
amount of protein and BCATm mRNA expression pattern as
well as the immunolocalization at cellular and subcellular
Correspondence to A. R. Tovar. Departamento de Fisiologı
´
adela
Nutricio
´
n, Instituto Nacional de Ciencias Me
´
dicas Nutricio
´
n ‘Salvador
Zubira
´
n’, Me
´
xico DF 14300, Me
´
xico. Fax: 1 525 6551076,
Tel.: 1 525 5731200 ext. 2801/2802,
E-mail:
Enzymes: branched chain amino acid aminotransferase (BCAT, EC
2.6.1.42).
(Received 25 June 2001, revised 27 September 2001, accepted
28 September 2001)

Abbreviations: BCAT, branched chain amino-acid aminotransferase;
BCATm, mitochondrial BCAT; BCAA, branched chain amino acids;
BCATc, cytosolic BCAT; BCODC, branched chain 2-oxo acid
dehydrogenase complex.
Eur. J. Biochem. 268, 6132–6139 (2001) q FEBS 2001
level of fetal and adult liver rats. The results show a new
localization of BCATm in the nuclei of fetal hepatocytes and
the presence of an active and inactive form of the BCATm in
fetal and adult liver, respectively.
MATERIALS AND METHODS
Fetal livers
Wistar rats of 17- and 19-days gestation were used.
Gestational age was determined by vaginal smear to detect
spermatozoa. Fetal livers were removed immediately,
pooled and then divided in to aliquots for RNA extraction,
Western blot analysis, BCAT enzyme assay and immuno-
histochemical studies. Heart and kidney were also processed
for comparison purposes. Samples for RNA extraction were
frozen in liquid nitrogen. This study was approved by the
Committee of Animal Research of the Instituto Nacional de
Ciencias Me
´
dicas y Nutricio
´
n ‘Salvador Zubira
´
n’, Me
´
xico.
Preparation of the supernatant fraction for BCAT assay

A sample of liver, kidney or heart was suspended in buffer
(4 mL extraction buffer per gram tissue) containing 225 m
M
mannitol, 75 mM sucrose, 0.1 mM EDTA, 5 mM Mops and a
mix of protease inhibitors including 1 m
M EDTA, 1 mM
EGTA, 1 mM diisopropylfluorophosphate, 5 mM benzami-
dine, 5 m
M dithiothreitol, 10 mg
:
mL
21
leupeptin and 1%
Triton X-100. Supernatant fraction of fetal liver or heart was
obtained from a pool of 19–24 fetuses. The tissue
suspension was centrifuged at 30 000 g for 60 min at
4 8C. The supernatant was assayed for BCAT activity.
Isolation of nuclei
Nuclei were isolated as described [13]. Liver was
homogenated in 10 m
M Tris/HCl pH 7.5 containing 0.3 M
sucrose, 5 mM dithiothreitol and 0.05% triton X-100. After
centrifugation at 83 000 g for 45 min with an 70 Ti rotor at
4 8C through a cushion of 2.3
M sucrose, 2 mM MgCl
2
,
10 m
M Tris/HCl pH 7.5, nuclei were counted and suspended
at a concentration of 2 Â 10

7
in buffer containing 50%
glycerol, 2 m
M MgCl
2
. 0.1 mM EDTA, 50 mM Hepes
pH 7.5, 0.1 m
M phenylmethanesulfonyl fluoride, and were
stored at 280 8C until use. Electron-microscopic analysis
revealed no contamination with mitochondria or other
cytoplasmic materials.
Determination of branched-chain amino-acid
aminotransferase activity
BCAT activity was assayed in all the supernatants and nuclei
by the method described previously [4,14]. Activity was
measured at 37 8Cin50m
M potassium phosphate buffer
pH 7.8, which contained 50 m
M pyridoxal phosphate and
4g
:
L
21
Chaps. Fifty microliters of supernatant were added
to the assay, and the reaction was initiated by addition of a
mixture containing 1.0 m
M 2-oxo[1-
14
C]isocaproate/12 mM
isoleucine. The specific activity for 2-oxo[1-

14
C]isocaproate
was 3.3 Bq
:
nmol
21
. After 5 min the reaction was stopped
by addition of 500 mLof2
M sodium acetate pH 3.4. The
remaining 2-oxo[1-
14
C]isocaproate not transaminated was
chemically decarboxylated by adding 250 mL of 30%
hydrogen peroxide. A sample of 250 mL of the reaction
mixture was added to a scintillation vial. Then 10 mL of
liquid scintillation cocktail (BCS, Amersham) was added
and samples were counted (Wallac, Turku, Finland). Each
assay was performed in duplicate. A unit of activity was
defined as 1 mmol [1-
14
C]leucine formed per min at 37 8C.
BCAT specific activity was expressed as mU
:
mg protein
21
.
SDS/PAGE
SDS/PAGE was carried out according to Ausubel et al. [13]
in 10% gels using 40 mg of protein. Prior to electrophoresis,
all samples were boiled for 5 min in the presence of 4%

SDS, with 2% 2-mercaptoethanol. Premixed protein
molecular weight markers (low range) were used for
molecular mass determination (Boehringer Mannheim).
Immunoblotting
After electrophoresis the separated proteins were transferred
to poly(viynlidene difluoride) Western blotting membranes
(Boehringer Mannheim). The transfer was carried out in a
Transphor electrophoresis unit (Hoefer Scientific Instru-
ments) following the manufacturer’s instructions. The
poly(viynlidene difluoride) membranes were treated with
1.5% gelatin/1.5% albumin for 2 h at 37 8C and incubated
with anti-(rat BCATm) IgG (1 : 2500) for 1.5 h at room
temperature. Immunoreactive protein bands were visualized
using horseradish peroxidase-labeled goat anti-(rabbit Ig) Ig
(1 : 6000) after the oxidation of luminol as luminescent
substrate. The light emission was detected by a short
exposure to autoradiography film (ECL, Amersham Life
Science). Anti-(rat BCATm) IgG was obtained as described
previously [2]. Immunoblot analysis using mitochondrial or
tissue extracts from several tissues have shown only a single
band with a M
r
of 41 kDa, indicating that the antibody does
not cross react with other proteins and that it recognizes
BCATm epitopes [9,12].
Isolation of total RNA and Northern blot analysis
Total RNA was isolated from liver, heart, or placenta
according to Chomczynski and Sacchi [15]. For Northern
analysis, 20 mg RNA was subjected to electrophoresis in a
1.5% agarose gel containing 37% formaldehyde and

transferred to a nylon membrane filter Hybond-N
1
(Amersham) and cross-linked with a UV crosslinker
(Amersham). The probe was a 900-bp Pst1–Eco R1
fragment of rat BCATm cDNA cloned in pT7 Bluescript
[6] and labeled with deoxycytidine 5
0
[a-
32
P]dCTP
(3000 Ci
:
mmol
21
, Amersham) using the rediprime DNA
labeling system (Amersham). Filters were prehybridized
with rapid-hyb buffer (Amersham) at 65 8C for 45 min, and
then hybridized with the labeled probe for 2.5 h at 65 8C.
Membranes were washed once with 2 Â NaCl/Cit, 0.1%
SDS at room temperature for 20 min and then washed twice
with 0.1 Â NaCl/Cit, 0.1% SDS at 65 8C for 15 min each.
Digitized images were prepared and quantitation of
radioactivity in the bands was carried out by using the
Instant Imager electronic autoradiography system (Packard
Instruments). Membranes were also exposed to Extascan
film (Kodak) at 270 8C with an intensifying screen.
q FEBS 2001 Nuclear BCAT in fetal liver (Eur. J. Biochem. 268) 6133
RT/PCR
Reverse transcription (RT)/PCR was performed with 5 mg
RNA from rat fetal or adult liver, and kidney. Total RNA

was treated with DNAse (Life Technologies) and the RT
was primed with oligo(dT). Specific oligonucleotides
for BCATm used for PCR amplification were: forward
primer, 5
0
-ATCCAGCCCTTCCAGAACC-3
0
and reverse
primer, 5
0
-AGCCGATCCAACCAGGTAG-3
0
corresponding
to nucleotides 248–265 and 1208–1226 of rat heart
BCATm cDNA, respectively [6]. The reaction produced a
979-bp product when kidney or heart mRNA were used.
The oligonucleotides were synthesized with a
Beckman Oligo 1000 DNA synthesizer. The product was
sequenced by using dideoxinucleotide terminators
(Amersham).
5
0
and 3
0
RACE of liver BCATm cDNA
Total RNA from adult rat liver was isolated as described
above. 5
0
and 3
0

RACE was carried out according to
manufacturer instructions (Life Technologies). Gene
specific primers, including nested primers, were designed
based on the RT/PCR product of BCATm amplified from
adult rat liver. The external and nested gene specific reverse
primers for the 5
0
RACE amplification were: 5
0
-GGCGTA
CCTGCTTGTCTCTGC-3
0
and 5
0
-CAAAGAGCTGCAAT
GAGTAGT-3
0
corresponding to nucleotides 339–359 and
297–317 of rat heart BCATm cDNA, respectively. The
external and nested gene specific forward primers for the 3
0
RACE amplification were: 5
0
-CAGAAGGAGTTGAAGG
CTATT-3
0
and 5
0
-ACGGAACCAGTGCCCACGATT-3
0

cor-
responding to nucleotides 1127–1147 and 1152–1172 of
rat heart BCATm cDNA, respectively. Products of 5
0
and
3
0
RACE were sequenced by using dideoxinucleotide
terminators (Amersham).
Fig. 1. Developmental pattern of hepatic BCAT activity, amount of
BCATm protein and BCATm mRNA levels in the rat. (A) BCAT
activity in fetal and postnatal liver. Data are expressed as mean ^ SEM;
n ¼ 3 –19. (B) Western blot analysis of BCATm using anti-BCATm.
(C) Northern blot analysis of BCATm mRNA. All lanes contained liver
total RNA from at least three rats.
Fig. 2. Immunohistochemical localization of BCATm in fetal and
adult liver. (A) Fetal liver showed intense immuno-staining in the
cytoplasm (arrows) and nuclei (white asterisks), as well as in nuclei of
megacaryocytes located in the sinusoidal lumen (arrowheads). (B) In
contrast, adult liver showed mild immunostaining confined to the
cytoplasm of hepatocytes (both micrographs  400).
6134 N. Torres et al. (Eur. J. Biochem. 268) q FEBS 2001
Histology, immunohistochemistry and immunoelectronic
microscopy
For light microscopy, fetal and adult liver slices were fixed
by immersion in absolute ethanol for 24 h, embedded in
paraffin, sectioned at 5 mm, and stained with hematoxylin
and eosin for histological analysis. Imunohistochemical
detection of the BCATm was performed with the rabbit anti-
(rat BCATm) IgG fraction. Before incubation with the

primary antibody, the endogenous peroxidase activity was
quenched with 0.03% H
2
O
2
in absolute methanol; liver
sections were then incubated with the primary antibody
diluted 1 : 500 in NaCl/P
i
overnight at 4 8C. Bound
antibodies were detected with goat anti-(rabbit IgG) Ig
labeled with peroxidase diluted 1 : 100 in NaCl/P
i
and
diaminobenzidine. For negative controls tissue was
incubated with primary antibody previously pre-adsorbed
with purified enzyme.
For immunoelectron microscopy studies, small tissue
fragments of fetal and adult liver were fixed by immersion in
4% paraformaldehyde dissolved in Sorensen’s buffer pH 7.4
for 2 h at 4 8C. After rinsing, free aldehyde groups were
blocked in 0.5
M NH
4
Cl in NaCl/P
i
for 1 h. Tissue samples
were dehydrated in graded ethyl alcohols and embedded in
LR-White hydrosoluble resin. The same fixation and
embedding procedure was used for nuclei isolated from

liver by differential ultracentrifugation. Thin sections
(between 70 and 90 nm) were placed on nickel grids; the
grids were then incubated with the rabbit anti-(rat BCATm)
IgG fraction diluted 1 : 100 in NaCl/P
i
with 1% BSA and
0.5% Tween. After rinsing repeatedly with NaCl/P
i
, the
grids were incubated with goat anti-(rabbit IgG) Ig
conjugated to 5 nm gold particles diluted 1 : 20 in the
same buffer. The grids were stained with uranium salts and
examined in a Zeiss EM 10 electron microscope. For
quantification, electron micrographs at a magnification of
 40 000 were taken and the number of gold particles in 20
consecutive randomly chosen hepatocyte nuclei from fetal
and adult liver sections were counted.
Chemicals and reagents
L-[1-
14
C]Leucine and the nucleotide [a-
32
P]dCTP were
from Dupont NEN. The radioactive 2-oxo[1-
14
C]isocapro-
ate was synthesized from [1-
14
C]leucine as described
previously [16]. All other reagents were obtained from

commercial sources and were at least reagent grade.
RESULTS
Developmental pattern of liver BCAT activity
Maximal BCAT activity, 7.28 mU
:
mg protein
21
, occurred in
fetal liver at 17 days’ gestation. BCAT activity decreased
significantly after birth: by 68% and 94% at birth and 21
days after birth, respectively. Mean BCAT activity in adult
rat liver was 0.38 ^ 0.05 mU
:
mg protein
21
, approximately
2% of that in heart. After day 20 postnatal, liver BCAT
specific activity remained low, similar to the levels reported
in the literature (Fig. 1A).
Fig. 3. Subcellular localization of BCATm in fetal and adult liver
by immunoelectron microscopy. (A) Fetal hepatocytes showed
immunolabeling in mitochondria (m) and chromatin (c) associated to
the nuclear membrane (nm) (Â 50 000). (B) The same pattern of
nuclear immunolabeling was seen in nuclei isolated from fetal liver by
differential ultracentrifugation at 32 000 g. (C) At the structural level,
adult hepatocytes showed immunolabeling in mitochondria (m), and
occasional gold particles were found in cytoplasm (Â 50 000).
Bar ¼ 0.5 mm.
q FEBS 2001 Nuclear BCAT in fetal liver (Eur. J. Biochem. 268) 6135
Immunohistochemical localization of BCATm in fetal rat

liver
Fetal rat liver showed intense immunostaining to BCATm in
the cytoplasm and nuclei of hepatocytes, as well as in the
nuclei of blood cell precursors, particularly megakaryocytes
(Fig. 2A). At the structural level, immunogold particles
were seen in the mitochondria and nuclei of fetal
hepatocytes, particularly intense immunoreactivity was
found in chromatin associated with the nuclear membrane
(Fig. 3A). The nuclei of blood cell precursors showed
similar amounts and distribution of gold particles. In
contrast, adult liver showed only mild immunoreactivity
located exclusively in the cytoplasm of hepatocytes
(Fig. 2B). At the subcellular level, adult hepatocytes
showed immunolabeling in mitochondria and immuno-
reactivity in small vacuoles located near to the endoplasmic
reticulum. No labeling at all was observed in the nuclei
(Fig. 3C). The quantitative study revealed that fetal
hepatocyte nuclei had 309 ^ 34 gold particles, whereas
hepatocyte nuclei of adult liver had 13 ^ 6 gold particles
(P , 0.00007). The same immunolabeling pattern and similar
amount of gold particles was seen in isolated fetal liver
nuclei obtained by differential centrifugation (Fig. 3B).
Mitochondria were absent from these preparations.
Nuclear BCAT activity
This new localization of the BCATm in liver nuclei raised
the question of whether any of the enzyme activity was
actually associated with the nuclei of liver cells. To answer
this question, BCAT activity was measured in liver
supernatant and isolated nuclei from fetal and adult rats.
The specific activities of BCAT from 17-days’ gestation

fetal liver and adult liver are shown in Fig. 4. Fetal liver
nuclear BCAT specific activity was 0.65 ^ 0.08 mU
:
mg
protein
21
whereas it was undetectable in adult liver nuclei.
These results indicate that approximately 10% of the BCAT
activity in fetal liver is associated with the nuclei. BCAT
specific activity in fetal liver was 19-fold higher than in the
adult liver supernatant, indicating that liver has a high
capacity for transamination of BCAA during the fetal stage,
but that this is lost after birth. Furthermore, BCAT specific
activity in fetal liver is 35% of that found in adult heart
which is considered to be one of the organs with high BCAT
activity.
Western blot analysis of BCATm in fetal and adult rat liver
When equal amounts of protein (40 mg) were subjected to
SDS/PAGE and immunoblotting, a 41-kDa protein corre-
sponding to the active form of BCATm was detected in fetal
liver nuclei, fetal liver supernatant, heart supernatant and
kidney mitochondria. However, this protein was not found in
adult liver nuclei or adult liver supernatant, indicating that
the protein found in fetal liver nuclei was the same of that
found in kidney and heart. The BCATm antiserum
recognized a protein of < 43 kDa in addition to the
41-kDa protein in fetal liver supernatant. In adult liver
supernatant only a faint 43-kDa band was seen (Fig. 4).
Thus, the appearance of the 41-kDa protein on immunoblots
was always associated with the presence of the highly active

form of BCATm. These results suggest the existence of an
active and inactive form of the BCAT, and the develop-
mental changes in BCAT activity in rat liver coincided with
the appearance and disappearance of the 41-kDa BCATm
(Fig. 1B).
BCATm mRNA expression in fetal and adult rat liver
The expression of BCATm during fetal development in the
rat was examined by measuring BCATm mRNA abundance.
Northern blot analysis detected a band of 1.7 kb that
corresponds to the size of the mRNA reported for this
enzyme in rat heart [6]. However, the expression of BCATm
mRNA in liver was detectable by Northern blot analysis
only on days 17 and 19 of gestation but not after birth
(Fig. 1C). As we detected a protein of 43 kDa in adult liver
with BCATm antiserum by Western blotting, we considered
that the apparent absence of BCATm mRNA in adult rat
liver was perhaps associated with the low abundance of its
message, and that the Northern blot analysis was not
sensitive enough to detect it. A RT/PCR assay was carried
out using primers designed to amplify an internal sequence
of BCATm cDNA. A band of 979 bp was detected when
total RNA from adult liver, fetal liver or kidney were used,
indicating that the RNA that codes for the 43 kDa is
possibly derived from the BCATm gene (Fig. 5). Sequenc-
ing of the PCR product obtained from adult rat liver showed
Fig. 4. Western blot analysis and enzyme activity of BCATm in
different cell fractions. Cell fractions were obtained as described in
Materials and methods.
Fig. 5. Expression of BCATm mRNA in kidney and adult or fetal
liver. Total RNA was isolated from kidney and liver. cDNA was

obtained by reverse transcription, and BCATm cDNA was amplified by
PCR using primers specific for rat BCATm. The size of the product
obtained was 979 bp. b-actin was used as standard for mRNA integrity.
6136 N. Torres et al. (Eur. J. Biochem. 268) q FEBS 2001
100% homology with the sequence of BCATm heart cDNA.
Furthermore, 5
0
and 3
0
RACE amplified the end terminals of
rat liver cDNA, and a single band for each amplification was
obtained. The sequence of the products of both amplifica-
tions also showed 100% homology with rat heart BCATm
cDNA. These results suggest that the low abundance mRNA
for the 43-kDa protein is possibly derived from the same
gene that produces the 41-kDa protein.
Developmental pattern of heart BCAT
BCAT activity in heart showed a different developmental
pattern than that observed in liver. BCAT activity increased
significantly (P , 0.01) up to day 21 after birth, and it was
25% higher with respect to the activity at birth. On day 21,
the BCAT activity reached the values reported for this organ
in adults rats. Western blot and Northern blot analysis
followed a similar pattern (Fig. 6A, and C).
DISCUSSION
The activity of several hepatic amino-acid degrading
enzymes is absent or low during fetal life, increases rapidly
at birth, and reaches the activity level found in adults from
12 h to several days after birth [17–20]. On the contrary, the
activity of hepatic BCAT followed a different develop-

mental pattern. Fetal liver showed significant BCAT activity
and BCATm mRNA expression decayed immediately after
birth. Postnatal liver showed low BCAT activity and negligible
BCATm mRNA expression. This pattern is observed in only
liver, as BCAT activity, amount of protein and BCATm
mRNA expression was present in heart during the fetal stage
and increased progressively as a function of age (Fig. 6).
Furthermore, this developmental pattern was specific for
BCATm in liver as BCATc expression was not observed in
this organ (data not shown). Previous studies indicated that
BCAT activity in fetal liver was associated with hemato-
poietic cells. Our immunohistochemical results showed that
BCATm is located in the nuclei and mitochondria of fetal
hepatic and hematopoietic cells; however, the proportion of
the former is greater than the latter indicating that the
contribution of BCATm activity is associated mainly to
hepatic cells.
This is the first report showing that BCATm is localized
in two subcellular organelles, the mitochondria and the
nucleus in fetal liver. The majority of proteins have only one
cellular destination; however, there is a class of enzymes
called sorting isozymes that are produced by the same gene
and that have multiple destinations [21]. Some enzymes of
this class are found in mitochondria and the cytoplasm
[22,23], cytoplasm and nuclei [24], mitochondria, cyto-
plasm and nuclei [21], and mitochondria and nuclei [25]. It
has been established that the 27 amino-acid pre-sequence of
BCATm contains information to target this enzyme to the
mitochondria [6]. However, the nuclear import of proteins
from the cytoplasm depends in part on the presence of a

short stretch of cationic amino acids containing four to six
residues of lysine or arginine [26]. An examination of the
mature BCATm protein showed that it does not contain a
typical consensus sequence for its import to the nuclei.
However this protein contains two cationic rich stretches,
located between amino acids 80 and 90 (KAYKGR
DKQVR) and 290 and 299 (RKVTMKELKR) that may
contribute to the nuclear localization of the enzyme. Perhaps
BCATm protein is transported to the nuclei by a specific
importin [27] that is present only during fetal life.
The high expression of BCATm during fetal life and the
very low branched-chain 2-oxo acid dehydrogenase
complex activity in liver and heart [20] reduce the oxidation
Fig. 6. Developmental pattern of BCAT activity, amount of BCATm
protein and BCATm mRNA levels in rat heart. (A) BCAT activity in
fetal and postnatal heart. The results are expressed as mean ^ SEM,
n ¼ 4 –24. (B) Western blot analysis of BCATm using anti-BCATm.
(C) Northern blot analysis of BCATm mRNA. All lanes contained heart
total RNA from at least four different rats.
Fig. 7. Northern blot analysis of BCATc and BCATm in rat
placenta. Total RNA was isolated from placenta of rats on day 17 and
19 of gestation as described in Materials and methods. Blots were
hybridized with the 900 bp Pst1 Eco R1 fragment of rat BCATm cDNA
or the 1400 bp Eco R1 fragment of rat BCATc cDNA [35].
q FEBS 2001 Nuclear BCAT in fetal liver (Eur. J. Biochem. 268) 6137
of BCAA: there is no need life for the disposal of these
amino acids during fetal. Thus, transamination of branched
chain 2-oxo acids by BCATm may play a specific role in
BCAA conservation which can then be used in protein
synthesis [28] during gestation.

It is probable that BCATm plays an important role in the
reamination of branched chain 2-oxo acids because of an
increase in the concentration of glutamate in fetal liver at the
end of pregnancy [29]. These data agree with known
nitrogen conservation schemes in pregnancy and with the
important demands on amino-acid supply by fetal growth. In
this phase of fetal growth the placental amino-acid uptake is
considerable and seems to be higher than immediately
before birth [29]. An increasing capacity for glutamate
absorption by the developing placenta has been demon-
strated. This concentrative absorption of glutamate by the
developing placenta is critical for proper fetal development
[30]. As shown in Fig. 7 there is a high expression of
BCATc and BCATm isoenzymes in placenta that may
contribute to the transamination of BCAA to produce
glutamate and branched chain 2-oxo acids which can be
used by the fetus.
This situation is reversed after birth, the activity and
expression of the active form of liver BCATm decreases
dramatically after birth, whereas in other extrahepatic
tissues such as heart, BCATm activity and mRNA
expression increase. On the other hand, BCODC activity
increases dramatically in liver and heart during the suckling
period thus increasing the oxidative capacity of BCAA after
birth [20]. During postnatal life, we observed the appearance
of an inactive form of the BCATm in adult liver; therefore
BCAA are shuttled to extra-hepatic tissues in the adult rat
thus preventing their oxidation in liver [31]. The results
of this study suggest that there is no alternative splicing
of the BCATm gene; the sequence of the cDNA from

liver is the same as that of heart BCATm cDNA [6]. It
is possible that some step in the processing of the
BCATm protein is inactive in the adult liver, but is
active in fetal liver. Studies in our laboratory are in
progress to elucidate the mechanism of regulation of the
two forms in liver. At the present time, we cannot rule
out the possibility that the 43-kDa protein is responsible
for the low BCAT activity in liver, although it has been
proposed that BCAA are transaminated by the
asparagine aminotransferase in liver.
Although BCATm expression is unresponsive to dietary
protein or hormones (hydrocortisone and glucagon) in
extrahepatic tissues [4], conditions related to cell growth as
in fetal liver [11], growth of hepatocytes in culture [32], and
lactating mammary gland tissue [8,9] stimulates BCATm
activity and expression. There is evidence to support the role
of BCAT in cell growth. Two yeast proteins have been
shown to function as BCAT [33,34]; mutation of one of
these BCAT homologs produces a short G1 stage indicating
that this protein is involved in cell cycle regulation. On
the other hand, the mouse BCATc gene is highly
expressed early in embryogenesis and in several c-myc
based tumors. Thus, BCAT may play additional roles in
situations where high cell proliferation takes place and
perhaps the nuclear localization of BCATm in fetal liver
is involved in one of them. Further studies are required
to clarify the possible role of BCAT in situations of
accelerated cell proliferation.
ACKNOWLEDGEMENT
Financial support was from CONACYT 25637M (to A. R. T.), Me

´
xico.
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