Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid...

Ontogeny and subcellular localization of rat liver mitochondrialbranched chain amino-acid aminotransferase

Nimbe Torres1, Carolina Vargas1, Rogelio Hernandez-Pando2, Hector Orozco2, Susan M. Hutson3 andArmando R. Tovar1

1Departamento de Fisiologıa de la Nutricion, Instituto Nacional de Ciencias Medicas y Nutricion ‘Salvador Zubiran’, Mexico;2Departamento de Patologıa Experimental, Instituto Nacional de Ciencias Medicas y Nutricion ‘Salvador Zubiran’,Mexico; 3Department 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:mg21 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 proteinsynthesis. The initial enzymes for catabolism of the BCAAare regulated differently from other amino-acid degradingenzymes. The first step in the degradation of these aminoacids is a reversible transamination catalyzed by thebranched chain amino-acid aminotransferase (BCAT;EC 2.6.1.42). The products of this reaction are thecorresponding branched chain 2-oxo acids that can bereaminated to their corresponding amino acids [1], orirreversibly decarboxylated by the branched-chain 2-oxoacid dehydrogenase complex (BCODC) forming thecorresponding acyl CoA derivates. In mammals there aretwo BCAT isoenzymes, a mitochondrial (BCATm), and acytosolic (BCATc) form [2,3]. In the rat BCATm is thepredominant 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 inskeletal muscle and skin and negligible activity in adultliver. BCAT activity is accompanied by a similar pattern ofBCATm mRNA expression [4]. The cytosolic form isrestricted to brain, ovary and placenta [5]. BCATm cDNAencodes a polypeptide of 366 amino acids preceded by a pre-sequence of 27 amino acids with a molecular mass of themature protein of 41.2 kDa. The mature rat sequence is 82%and 95% identical to the human and murine BCATmrespectively [6] and 82% identical to sheep BCATm [7]. Incontrast with other hepatic amino-acid degrading enzymes[4], BCATm expression is not regulated by glucagon,glucocorticoids or high dietary protein. However, BCATmmRNA expression is highly induced in lactating mammarytissue and declines rapidly after weaning [8,9].

Previous studies have shown that fetal rat liver, in contrastwith adult rat liver, has BCAT activity but that this declinesrapidly after birth [10,11]. This decrease was associatedmainly with a decrease in the volume fraction ofhematopoietic cells in fetal rat liver [11] assuming that theenzyme activity was confined to only hematopoietic cellsand not to hepatic cells. However, studies with freshlyisolated hepatocytes from 18-days gestation fetal rats andfetal hepatocytes cultured for 2 days showed BCAT activity,indicating the possibility that not only the hematopoieticcells were responsible for BCAT activity but that fetalhepatocytes 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 aswell as the immunolocalization at cellular and subcellular

Correspondence to A. R. Tovar. Departamento de Fisiologıa de la

Nutricion, Instituto Nacional de Ciencias Medicas Nutricion ‘Salvador

Zubiran’, Mexico DF 14300, Mexico. Fax: 1 525 6551076,

Tel.: 1 525 5731200 ext. 2801/2802,

E-mail: [email protected]

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 newlocalization of BCATm in the nuclei of fetal hepatocytes andthe presence of an active and inactive form of the BCATm infetal and adult liver, respectively.

M A T E R I A L S A N D M E T H O D S

Fetal livers

Wistar rats of 17- and 19-days gestation were used.Gestational age was determined by vaginal smear to detectspermatozoa. 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 processedfor comparison purposes. Samples for RNA extraction werefrozen in liquid nitrogen. This study was approved by theCommittee of Animal Research of the Instituto Nacional deCiencias Medicas y Nutricion ‘Salvador Zubiran’, Mexico.

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 mM

mannitol, 75 mM sucrose, 0.1 mM EDTA, 5 mM Mops and amix of protease inhibitors including 1 mM EDTA, 1 mM

EGTA, 1 mM diisopropylfluorophosphate, 5 mM benzami-dine, 5 mM dithiothreitol, 10 mg:mL21 leupeptin and 1%Triton X-100. Supernatant fraction of fetal liver or heart wasobtained from a pool of 19–24 fetuses. The tissuesuspension was centrifuged at 30 000 g for 60 min at4 8C. The supernatant was assayed for BCAT activity.

Isolation of nuclei

Nuclei were isolated as described [13]. Liver washomogenated in 10 mM Tris/HCl pH 7.5 containing 0.3 M

sucrose, 5 mM dithiothreitol and 0.05% triton X-100. Aftercentrifugation at 83 000 g for 45 min with an 70 Ti rotor at4 8C through a cushion of 2.3 M sucrose, 2 mM MgCl2,10 mM Tris/HCl pH 7.5, nuclei were counted and suspendedat a concentration of 2 � 107 in buffer containing 50%glycerol, 2 mM MgCl2. 0.1 mM EDTA, 50 mM HepespH 7.5, 0.1 mM phenylmethanesulfonyl fluoride, and werestored at 280 8C until use. Electron-microscopic analysisrevealed no contamination with mitochondria or othercytoplasmic materials.

Determination of branched-chain amino-acidaminotransferase activity

BCAT activity was assayed in all the supernatants and nucleiby the method described previously [4,14]. Activity wasmeasured at 37 8C in 50 mM potassium phosphate bufferpH 7.8, which contained 50 mM pyridoxal phosphate and4 g:L21 Chaps. Fifty microliters of supernatant were addedto the assay, and the reaction was initiated by addition of amixture containing 1.0 mM 2-oxo[1-14C]isocaproate/12 mM

isoleucine. The specific activity for 2-oxo[1-14C]isocaproatewas 3.3 Bq:nmol21. After 5 min the reaction was stoppedby addition of 500 mL of 2 M sodium acetate pH 3.4. Theremaining 2-oxo[1-14C]isocaproate not transaminated waschemically decarboxylated by adding 250 mL of 30%

hydrogen peroxide. A sample of 250 mL of the reactionmixture was added to a scintillation vial. Then 10 mL ofliquid scintillation cocktail (BCS, Amersham) was addedand samples were counted (Wallac, Turku, Finland). Eachassay was performed in duplicate. A unit of activity wasdefined as 1 mmol [1-14C]leucine formed per min at 37 8C.BCAT specific activity was expressed as mU:mg protein21.

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 proteinmolecular weight markers (low range) were used formolecular mass determination (Boehringer Mannheim).

Immunoblotting

After electrophoresis the separated proteins were transferredto poly(viynlidene difluoride) Western blotting membranes(Boehringer Mannheim). The transfer was carried out in aTransphor electrophoresis unit (Hoefer Scientific Instru-ments) following the manufacturer’s instructions. Thepoly(viynlidene difluoride) membranes were treated with1.5% gelatin/1.5% albumin for 2 h at 37 8C and incubatedwith anti-(rat BCATm) IgG (1 : 2500) for 1.5 h at roomtemperature. Immunoreactive protein bands were visualizedusing horseradish peroxidase-labeled goat anti-(rabbit Ig) Ig(1 : 6000) after the oxidation of luminol as luminescentsubstrate. The light emission was detected by a shortexposure to autoradiography film (ECL, Amersham LifeScience). Anti-(rat BCATm) IgG was obtained as describedpreviously [2]. Immunoblot analysis using mitochondrial ortissue extracts from several tissues have shown only a singleband with a Mr of 41 kDa, indicating that the antibody doesnot cross react with other proteins and that it recognizesBCATm epitopes [9,12].

Isolation of total RNA and Northern blot analysis

Total RNA was isolated from liver, heart, or placentaaccording to Chomczynski and Sacchi [15]. For Northernanalysis, 20 mg RNA was subjected to electrophoresis in a1.5% agarose gel containing 37% formaldehyde andtransferred to a nylon membrane filter Hybond-N1

(Amersham) and cross-linked with a UV crosslinker(Amersham). The probe was a 900-bp Pst1–Eco R1fragment of rat BCATm cDNA cloned in pT7 Bluescript[6] and labeled with deoxycytidine 50[a-32P]dCTP(3000 Ci:mmol21, Amersham) using the rediprime DNAlabeling system (Amersham). Filters were prehybridizedwith rapid-hyb buffer (Amersham) at 65 8C for 45 min, andthen hybridized with the labeled probe for 2.5 h at 65 8C.Membranes were washed once with 2 � NaCl/Cit, 0.1%SDS at room temperature for 20 min and then washed twicewith 0.1 � NaCl/Cit, 0.1% SDS at 65 8C for 15 min each.Digitized images were prepared and quantitation ofradioactivity in the bands was carried out by using theInstant Imager electronic autoradiography system (PackardInstruments). Membranes were also exposed to Extascanfilm (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 mgRNA from rat fetal or adult liver, and kidney. Total RNAwas treated with DNAse (Life Technologies) and the RTwas primed with oligo(dT). Specific oligonucleotidesfor BCATm used for PCR amplification were: forwardprimer, 50-ATCCAGCCCTTCCAGAACC-30 and reverseprimer, 50-AGCCGATCCAACCAGGTAG-30 correspondingto nucleotides 248–265 and 1208–1226 of rat heartBCATm cDNA, respectively [6]. The reaction produced a979-bp product when kidney or heart mRNA were used.The oligonucleotides were synthesized with aBeckman Oligo 1000 DNA synthesizer. The product wassequenced by using dideoxinucleotide terminators(Amersham).

50 and 30 RACE of liver BCATm cDNA

Total RNA from adult rat liver was isolated as describedabove. 50 and 30 RACE was carried out according tomanufacturer instructions (Life Technologies). Genespecific primers, including nested primers, were designedbased on the RT/PCR product of BCATm amplified fromadult rat liver. The external and nested gene specific reverseprimers for the 50 RACE amplification were: 50-GGCGTACCTGCTTGTCTCTGC-30 and 50-CAAAGAGCTGCAATGAGTAGT-30 corresponding to nucleotides 339–359 and297–317 of rat heart BCATm cDNA, respectively. The

external and nested gene specific forward primers for the 30

RACE amplification were: 50-CAGAAGGAGTTGAAGGCTATT-30 and 50-ACGGAACCAGTGCCCACGATT-30 cor-responding to nucleotides 1127–1147 and 1152–1172 ofrat heart BCATm cDNA, respectively. Products of 50 and30 RACE were sequenced by using dideoxinucleotideterminators (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 immunoelectronicmicroscopy

For light microscopy, fetal and adult liver slices were fixedby immersion in absolute ethanol for 24 h, embedded inparaffin, sectioned at 5 mm, and stained with hematoxylinand eosin for histological analysis. Imunohistochemicaldetection of the BCATm was performed with the rabbit anti-(rat BCATm) IgG fraction. Before incubation with theprimary antibody, the endogenous peroxidase activity wasquenched with 0.03% H2O2 in absolute methanol; liversections were then incubated with the primary antibodydiluted 1 : 500 in NaCl/Pi overnight at 4 8C. Boundantibodies were detected with goat anti-(rabbit IgG) Iglabeled with peroxidase diluted 1 : 100 in NaCl/Pi anddiaminobenzidine. For negative controls tissue wasincubated with primary antibody previously pre-adsorbedwith purified enzyme.

For immunoelectron microscopy studies, small tissuefragments of fetal and adult liver were fixed by immersion in4% paraformaldehyde dissolved in Sorensen’s buffer pH 7.4for 2 h at 4 8C. After rinsing, free aldehyde groups wereblocked in 0.5 M NH4Cl in NaCl/Pi for 1 h. Tissue sampleswere dehydrated in graded ethyl alcohols and embedded inLR-White hydrosoluble resin. The same fixation andembedding procedure was used for nuclei isolated fromliver by differential ultracentrifugation. Thin sections(between 70 and 90 nm) were placed on nickel grids; thegrids were then incubated with the rabbit anti-(rat BCATm)IgG fraction diluted 1 : 100 in NaCl/Pi with 1% BSA and0.5% Tween. After rinsing repeatedly with NaCl/Pi, thegrids were incubated with goat anti-(rabbit IgG) Igconjugated to 5 nm gold particles diluted 1 : 20 in thesame buffer. The grids were stained with uranium salts andexamined in a Zeiss EM 10 electron microscope. Forquantification, electron micrographs at a magnification of� 40 000 were taken and the number of gold particles in 20consecutive randomly chosen hepatocyte nuclei from fetaland adult liver sections were counted.

Chemicals and reagents

L-[1-14C]Leucine and the nucleotide [a-32P]dCTP werefrom Dupont NEN. The radioactive 2-oxo[1-14C]isocapro-ate was synthesized from [1-14C]leucine as describedpreviously [16]. All other reagents were obtained fromcommercial sources and were at least reagent grade.

R E S U L T S

Developmental pattern of liver BCAT activity

Maximal BCAT activity, 7.28 mU:mg protein21, occurred infetal liver at 17 days’ gestation. BCAT activity decreased

significantly after birth: by 68% and 94% at birth and 21days after birth, respectively. Mean BCAT activity in adultrat liver was 0.38 ^ 0.05 mU:mg protein21, approximately2% of that in heart. After day 20 postnatal, liver BCATspecific activity remained low, similar to the levels reportedin 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) (� 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 (� 50 000).

Bar ¼ 0.5 mm.


Immunohistochemical localization of BCATm in fetal ratliver

Fetal rat liver showed intense immunostaining to BCATm inthe cytoplasm and nuclei of hepatocytes, as well as in thenuclei of blood cell precursors, particularly megakaryocytes(Fig. 2A). At the structural level, immunogold particleswere seen in the mitochondria and nuclei of fetalhepatocytes, particularly intense immunoreactivity wasfound in chromatin associated with the nuclear membrane(Fig. 3A). The nuclei of blood cell precursors showedsimilar amounts and distribution of gold particles. Incontrast, adult liver showed only mild immunoreactivitylocated exclusively in the cytoplasm of hepatocytes(Fig. 2B). At the subcellular level, adult hepatocytesshowed immunolabeling in mitochondria and immuno-reactivity in small vacuoles located near to the endoplasmicreticulum. No labeling at all was observed in the nuclei(Fig. 3C). The quantitative study revealed that fetalhepatocyte nuclei had 309 ^ 34 gold particles, whereashepatocyte nuclei of adult liver had 13 ^ 6 gold particles(P , 0.00007). The same immunolabeling pattern and similaramount of gold particles was seen in isolated fetal livernuclei obtained by differential centrifugation (Fig. 3B).Mitochondria were absent from these preparations.

Nuclear BCAT activity

This new localization of the BCATm in liver nuclei raisedthe question of whether any of the enzyme activity wasactually associated with the nuclei of liver cells. To answerthis question, BCAT activity was measured in liversupernatant and isolated nuclei from fetal and adult rats.The specific activities of BCAT from 17-days’ gestationfetal liver and adult liver are shown in Fig. 4. Fetal livernuclear BCAT specific activity was 0.65 ^ 0.08 mU:mgprotein21 whereas it was undetectable in adult liver nuclei.These results indicate that approximately 10% of the BCATactivity in fetal liver is associated with the nuclei. BCATspecific activity in fetal liver was 19-fold higher than in theadult liver supernatant, indicating that liver has a highcapacity for transamination of BCAA during the fetal stage,but that this is lost after birth. Furthermore, BCAT specificactivity in fetal liver is 35% of that found in adult heartwhich is considered to be one of the organs with high BCATactivity.

Western blot analysis of BCATm in fetal and adult rat liver

When equal amounts of protein (40 mg) were subjected toSDS/PAGE and immunoblotting, a 41-kDa protein corre-sponding to the active form of BCATm was detected in fetalliver nuclei, fetal liver supernatant, heart supernatant andkidney mitochondria. However, this protein was not found inadult liver nuclei or adult liver supernatant, indicating thatthe protein found in fetal liver nuclei was the same of thatfound in kidney and heart. The BCATm antiserumrecognized a protein of < 43 kDa in addition to the41-kDa protein in fetal liver supernatant. In adult liversupernatant only a faint 43-kDa band was seen (Fig. 4).Thus, the appearance of the 41-kDa protein on immunoblotswas always associated with the presence of the highly activeform 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 withthe 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 therat was examined by measuring BCATm mRNA abundance.Northern blot analysis detected a band of 1.7 kb thatcorresponds to the size of the mRNA reported for thisenzyme in rat heart [6]. However, the expression of BCATmmRNA in liver was detectable by Northern blot analysisonly on days 17 and 19 of gestation but not after birth(Fig. 1C). As we detected a protein of 43 kDa in adult liverwith BCATm antiserum by Western blotting, we consideredthat the apparent absence of BCATm mRNA in adult ratliver was perhaps associated with the low abundance of itsmessage, and that the Northern blot analysis was notsensitive enough to detect it. A RT/PCR assay was carriedout using primers designed to amplify an internal sequenceof BCATm cDNA. A band of 979 bp was detected whentotal RNA from adult liver, fetal liver or kidney were used,indicating that the RNA that codes for the 43 kDa ispossibly 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.


100% homology with the sequence of BCATm heart cDNA.Furthermore, 50 and 30 RACE amplified the end terminals ofrat liver cDNA, and a single band for each amplification wasobtained. The sequence of the products of both amplifica-tions also showed 100% homology with rat heart BCATmcDNA. These results suggest that the low abundance mRNAfor the 43-kDa protein is possibly derived from the samegene that produces the 41-kDa protein.

Developmental pattern of heart BCAT

BCAT activity in heart showed a different developmentalpattern than that observed in liver. BCAT activity increasedsignificantly (P , 0.01) up to day 21 after birth, and it was25% higher with respect to the activity at birth. On day 21,the BCAT activity reached the values reported for this organin adults rats. Western blot and Northern blot analysisfollowed a similar pattern (Fig. 6A, and C).

D I S C U S S I O N

The activity of several hepatic amino-acid degradingenzymes is absent or low during fetal life, increases rapidlyat birth, and reaches the activity level found in adults from12 h to several days after birth [17–20]. On the contrary, theactivity of hepatic BCAT followed a different develop-mental pattern. Fetal liver showed significant BCAT activityand BCATm mRNA expression decayed immediately afterbirth. Postnatal liver showed low BCATactivity and negligible

BCATm mRNA expression. This pattern is observed in onlyliver, as BCAT activity, amount of protein and BCATmmRNA expression was present in heart during the fetal stageand increased progressively as a function of age (Fig. 6).Furthermore, this developmental pattern was specific forBCATm in liver as BCATc expression was not observed inthis organ (data not shown). Previous studies indicated thatBCAT activity in fetal liver was associated with hemato-poietic cells. Our immunohistochemical results showed thatBCATm is located in the nuclei and mitochondria of fetalhepatic and hematopoietic cells; however, the proportion ofthe former is greater than the latter indicating that thecontribution of BCATm activity is associated mainly tohepatic cells.

This is the first report showing that BCATm is localizedin two subcellular organelles, the mitochondria and thenucleus in fetal liver. The majority of proteins have only onecellular destination; however, there is a class of enzymescalled sorting isozymes that are produced by the same geneand that have multiple destinations [21]. Some enzymes ofthis 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]. Ithas been established that the 27 amino-acid pre-sequence ofBCATm contains information to target this enzyme to themitochondria [6]. However, the nuclear import of proteinsfrom the cytoplasm depends in part on the presence of ashort stretch of cationic amino acids containing four to sixresidues of lysine or arginine [26]. An examination of themature BCATm protein showed that it does not contain atypical consensus sequence for its import to the nuclei.However this protein contains two cationic rich stretches,located between amino acids 80 and 90 (KAYKGRDKQVR) and 290 and 299 (RKVTMKELKR) that maycontribute to the nuclear localization of the enzyme. PerhapsBCATm protein is transported to the nuclei by a specificimportin [27] that is present only during fetal life.

The high expression of BCATm during fetal life and thevery low branched-chain 2-oxo acid dehydrogenasecomplex activity in liver and heart [20] reduce the oxidation

Fig. 6. Developmental pattern of BCATactivity, 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].


of BCAA: there is no need life for the disposal of theseamino acids during fetal. Thus, transamination of branchedchain 2-oxo acids by BCATm may play a specific role inBCAA conservation which can then be used in proteinsynthesis [28] during gestation.

It is probable that BCATm plays an important role in thereamination of branched chain 2-oxo acids because of anincrease in the concentration of glutamate in fetal liver at theend of pregnancy [29]. These data agree with knownnitrogen conservation schemes in pregnancy and with theimportant demands on amino-acid supply by fetal growth. Inthis phase of fetal growth the placental amino-acid uptake isconsiderable and seems to be higher than immediatelybefore birth [29]. An increasing capacity for glutamateabsorption by the developing placenta has been demon-strated. This concentrative absorption of glutamate by thedeveloping placenta is critical for proper fetal development[30]. As shown in Fig. 7 there is a high expression ofBCATc and BCATm isoenzymes in placenta that maycontribute to the transamination of BCAA to produceglutamate and branched chain 2-oxo acids which can beused by the fetus.

This situation is reversed after birth, the activity andexpression of the active form of liver BCATm decreasesdramatically after birth, whereas in other extrahepatictissues such as heart, BCATm activity and mRNAexpression increase. On the other hand, BCODC activityincreases dramatically in liver and heart during the sucklingperiod thus increasing the oxidative capacity of BCAA afterbirth [20]. During postnatal life, we observed the appearanceof an inactive form of the BCATm in adult liver; thereforeBCAA are shuttled to extra-hepatic tissues in the adult ratthus preventing their oxidation in liver [31]. The resultsof this study suggest that there is no alternative splicingof the BCATm gene; the sequence of the cDNA fromliver is the same as that of heart BCATm cDNA [6]. Itis possible that some step in the processing of theBCATm protein is inactive in the adult liver, but isactive in fetal liver. Studies in our laboratory are inprogress to elucidate the mechanism of regulation of thetwo forms in liver. At the present time, we cannot ruleout the possibility that the 43-kDa protein is responsiblefor the low BCAT activity in liver, although it has beenproposed that BCAA are transaminated by theasparagine aminotransferase in liver.

Although BCATm expression is unresponsive to dietaryprotein or hormones (hydrocortisone and glucagon) inextrahepatic tissues [4], conditions related to cell growth asin fetal liver [11], growth of hepatocytes in culture [32], andlactating mammary gland tissue [8,9] stimulates BCATmactivity and expression. There is evidence to support the roleof BCAT in cell growth. Two yeast proteins have beenshown to function as BCAT [33,34]; mutation of one ofthese BCAT homologs produces a short G1 stage indicatingthat this protein is involved in cell cycle regulation. Onthe other hand, the mouse BCATc gene is highlyexpressed early in embryogenesis and in several c-mycbased tumors. Thus, BCAT may play additional roles insituations where high cell proliferation takes place andperhaps the nuclear localization of BCATm in fetal liveris involved in one of them. Further studies are requiredto clarify the possible role of BCAT in situations ofaccelerated cell proliferation.

A C K N O W L E D G E M E N T

Financial support was from CONACYT 25637M (to A. R. T.), Mexico.

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