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: artovar@quetzal.innsz.mx 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  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  NaCl/Cit, 0.1% SDS at room temperature for 20 min and then washed twice with 0.1  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) ( 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. 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. REFERENCES 1. Torres, N., Tovar, A.R. & Harper, A.E. (1993) Metabolism of valine in rat skeletal muscle mitochondria. J. Nutr. Biochem. 4, 681–689. 2. Wallin, R., Hall, T.R. & Hutson, S.M. (1990) Purification of branched chain aminotransferase. J. Biol. Chem. 265, 6019–6024. 3. Hall, T.R., Wallin, R., Reinhart, G.D. & Hutson, S.M. (1993) Branched chain aminotransferase isoenzymes. J. Biol. Chem. 268, 3092–3098. 4. Torres, N., Lo ´ pez, G., DeSantiago, S., Hutson, S.M. & Tovar, A.R. (1998) Dietary protein level regulates expression of the mitochondrial branched-chain aminotransferase in rats. J. Nutr. 128, 1368– 1375. 5. Ichihara, A. (1985) Aminotransferases of branched-chain amino acids. In Transaminases (Christen, P. & Metzler, D.E., eds), pp. 430– 500. John Wiley & Sons, New York. 6. Bledsoe, R.K., Dawson, P.A. & Hutson, S.M. (1997) Cloning of the rat and human mitochondrial branched chain aminotransferases (BCATm). Biochim. Biophys. Acta. 1339, 9– 13. 7. Faure, M., Glomot, F., Bledsoe, R., Hutson, S. & Papet, I. (1999) Purification and cloning of the mitochondrial branched-chain amino acid aminotransferase from sheep placenta. Eur. J. Biochem. 259, 104– 111. 8. De Santiago, S., Torres, N., Suryawan, A., Tovar, A.R. & Hutson, S.M. (1998) Regulation of branched-chain amino acid metabolism in the lactating rat. J. Nutr. 128, 1165–1171. 9. Tovar, A.R., Becerril, E., Herna ´ ndez-Pando, R., Lo ´ pez, G., Suryawan, A., DeSantiago, S., Hutson, S.M. & Torres, N. (2001) Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland. Am. J. Physiol. Endocrinol. Metab. 280, E480–E488. 10. Ichihara, A. (1975) Isozyme patterns of branched-chain amino acid transaminase during cellular differentiation and carcinogenesis. In Carcinofetal Proteins: Biology and Chemistry (Hirai, H. &Alpert, E., eds), pp. 347– 359. New York Academy of Sciences, New York. 11. Kadowaki, H. & Knox, W.E. (1982) Cytosolic and mitochondrial isoenzymes of branched-chain amino acid aminotransferase during development of the rat. Biochem. J. 202, 777– 783. 12. Hutson, S., Wallin, R. & Hall, T.R. (1992) Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues. J. Biol. Chem. 267, 15681–15686. 13. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K. (1994). Current Protocols in Molecular Biology. Wiley, New York. 14. Hutson, S.M. (1988) Subcellular distribution of branched-chain aminotransferase activity in rat tissues. J. Nutr. 118, 1475–1481. 15. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. 16. Ru ¨ diger, H.W., Langenbeck, U. & Goedde, H.W. (1972) A simplified method for the preparation of 14 C-labelled branched- chain a-oxo acids. Biochem. J. 126, 445– 446. 17. Feigelson, M. (1973) Studies on the postnatal development of responses of hepatic histidase to endocrine control. Biochim. Biophys. Acta. 304, 669–685. 18. Kang-Lee, Y.A. & Harper, A.E. (1975) Effect of maternal protein deprivation on enzymatic development in newborn rats. Proc. Soc. Exp. Biol. Med. 149, 610– 614. 19. Grogan, C.K., Janas, L.M., Hendrix, M.K., Layman, D.K. & Picciano, M.F. (1988) Impact of nutrition on postnatal development of serine-threonine dehydratase and branched-chain keto acid dehydrogenase in the rat. Biol. Neonate 54, 224–231. 20. Zhao, Y., Denne, S. & Harris, R.A. (1993) Developmental pattern 6138 N. Torres et al. (Eur. J. Biochem. 268) q FEBS 2001 of branched-chain 2-oxo acid dehydrogenase complex in rat liver and heart. Biochem. J. 290, 395–399. 21. Gillman, E.C., Slusher, L.B., Martin, N.C. & Hopper, A.K. (1991) MOD5 translation initiation sites determine N6-isopentenyl adenosine modification of mitochondrial and cytoplasmic tRNA. Mol. Cell. Biol. 11, 2382– 2390. 22. Doonan, S., Barra, D. & Bossa, F. (1984) Structural and genetic relationship between cytosolic and mitochondrial isoenzymes. Int. J. Biochem. 16, 1193–1199. 23. Suzuki, T., Sato, M., Yoshida, T. & Tuboi, S. (1989) Rat liver mitochondria and cytosolic fumarases with identical amino acid sequences are enconded from a single gene. J. Biol. Chem. 264, 2581–2586. 24. Mainwaring, G.W., Foster, J.R. & Green, T. (1998) Nuclear and cellular immunolocalization of theta-class glutathione S-transfer- ase GSTT1-1 in the liver and lung of the mouse. Biochem. J. 329, 431–432. 25. Rose, A.M., Joyce, P.B., Hopper, A.K. & Martin, N.C. (1992) Separate information required for nuclear and subcellular localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei. Mol. Cell. Biol. 12, 5652 –5658. 26. Chelsky, D., Ralph, R. & Jonak, G. (1989) Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol. Cell. Biol. 9, 2487–2492. 27. Gorlich, D. & Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell. Dev. Biol. 15, 607–660. 28. Shiota, T., Yagi, M. & Walser, M. (1989) Utilization for protein synthesis in individual rat organs of extracellular 2 ketoisocaproate relative to utilization of extracellular leucine. Metabolism 38, 612–618. 29. Palou, A., Arola, L. & Alemany, M. (1977) Plasma amino acid concentrations in pregnant rats and in 21-day foetuses. Biochem. J. 166, 49– 55. 30. Matthews, J.C., Beveridge, M.J., Malandro, M.S., Rothstein, J.D., Campbell-Thompson, M., Verlander, J.W., Kilberg, M.S. & Novak, D.A. (1998) Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta. Am. J. Physiol. 274, C603– C614. 31. Harper, A.E., Miller, R.H. & Block, K.P. (1984) Branched-chain amino acid metabolism. Ann. Rev. Nutr. 4, 409–454. 32. Ichihara, A., Noda, C. & Tanaka, K. (1981) Oxidation of branched chain amino acids with special reference to their transaminase. In Metabolism and Clinical Implications of Branched Chain Amino and Ketoacids (Walser, M. &Williamson, J.R., eds), pp. 227–231. Elsevier/North Holland, Amsterdam, the Netherlands. 33. Kispal, G., Steiner, H., Court, D., Rolinski, B. & Lill, R. (1996) Mitochondrial and cytosolic branched-chain amino acid transam- inases fron yeast, homologs of the myc oncogene-regulated Eca39 protein. J. Biol. Chem. 271, 24458–24464. 34. Eden, A., Simchen, G. & Benvenisty, N. (1996) Two yeast homologs of ECA39, a target for c-Myc regulation, code for cytosolic and mitochondrial branched-chain amino acid amono- transferases. J. Biol. Chem. 271, 20242–20245. 35. Hutson, S.M., Bledsoe, R.K., Hall, T.R. & Dawson, P.A. (1995) Cloning and expression of the mammalian cytosolic branched chain aminotransferase isoenzyme. J. Biol. Chem. 270, 30344–30352. q FEBS 2001 Nuclear BCAT in fetal liver (Eur. J. Biochem. 268) 6139 . 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. 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. York Academy of Sciences, New York. 11. Kadowaki, H. & Knox, W.E. (1982) Cytosolic and mitochondrial isoenzymes of branched- chain amino acid aminotransferase during development of the rat. Biochem.