Branched-chain amino acid catabolism: unique segregation of pathway enzymes in organ systems and peripheral nerves

We have examined the localization of the first two enzymes in the branched-chain amino acid (BCAA) catabolic pathway: the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) and the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Antibodies specific for BCATm or BCATc were used to immunolocalize the respective isozymes in cryosections of rat tissues. BCATm was expressed in secretory epithelia throughout the digestive tract, with the most intense expression in the stomach. BCATm was also strongly expressed in secretory cells of the exocrine pancreas, uterus, and testis, as well as in the transporting epithelium of convoluted tubules in kidney. In muscle, BCATm was located in myofibrils. Liver, as predicted, was not immunoreactive for BCATm. Unexpectedly, BCATc was localized in elements of the autonomic innervation of the digestive tract, as well as in axons in the sciatic nerve. The distributions of BCATc and BCATm did not overlap. BCATm-expressing cells also expressed the second enzyme of the BCAA catabolic pathway, BCKD. In selected monkey and human tissues examined by immunoblot and/or immunohistochemistry, BCATm and BCATc were distributed in patterns very similar to those found in the rat. The results show that BCATm is in a position to regulate BCAA availability as protein precursors and anabolic signals in secretory portions of the digestive and other organ systems. The unique expression of BCATc in neurons of the peripheral nervous system, without coexpression of BCKD, raises new questions about the physiological function of this BCAT isozyme.     

Role of Branched-Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism

Abstract: Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α-ketoglutarate to form the branched-chain α-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-¹⁴C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.     

Widespread neuronal expression of branched-chain aminotransferase in the CNS: implications for leucine/glutamate metabolism and for signaling by amino acids

Transamination of the branched-chain amino acids produces glutamate and branched-chain alpha-ketoacids. The reaction is catalyzed by branched-chain aminotransferase (BCAT), of which there are cytosolic and mitochondrial isoforms (BCATc and BCATm). BCATc accounts for 70% of brain BCAT activity, and contributes at least 30% of the nitrogen required for glutamate synthesis. In previous work, we showed that BCATc is present in the processes of glutamatergic neurons and in cell bodies of GABAergic neurons in hippocampus and cerebellum. Here we show that this metabolic enzyme is expressed throughout the brain and spinal cord, with distinct differences in regional and intracellular patterns of expression. In the cerebral cortex, BCATc is present in GABAergic interneurons and in pyramidal cell axons and proximal dendrites. Axonal labeling for BCATc continues into the corpus callosum and internal capsule. BCATc is expressed by GABAergic neurons in the basal ganglia and by glutamatergic neurons in the hypothalamus, midbrain, brainstem, and dorsal root ganglia. BCATc is also expressed in hypothalamic peptidergic neurons, brainstem serotoninergic neurons, and spinal cord motor neurons. The results indicate that BCATc accumulates in neuronal cell bodies in some regions, while elsewhere it is exported to axons and nerve terminals. The enzyme is in a position to influence pools of glutamate in a variety of neuronal types. BCATc may also provide neurons with sensitivity to nutrient-derived BCAAs, which may be important in regions that control feeding behavior, such as the arcuate nucleus of the hypothalamus, where neurons express high levels of BCATc.     

Expression of cytosolic branched chain aminotransferase (BCATc) mRNA in the developing mouse brain

Branched-chain aminotransferase (BCAT) catalyzes the transamination of essential branched-chain amino acids (BCAAs: leucine, isoleucine and valine) with alpha-ketoglutarate. Through this reaction, BCAAs provide nitrogen for the synthesis of glutamate, the predominant excitatory neurotransmitter. Two BCAT isoforms have been identified: one cytosolic (BCATc) and one mitochondrial (BCATm). In adult rodents, BCATc is expressed in a wide variety of structures of the central nervous system (CNS), in neurons. So far, no data were available about the expression of BCATc in the developing CNS. Here, we analyse the expression profile of BCATc mRNA in the mouse brain from embryonic day 12.5 to adult age. BCATc mRNA gradually appears in different brain regions starting from early stages of neural development, and is maintained until adulthood. BCATc mRNA is predominantly present in the cerebral cortex, hippocampus, thalamus, ventral midbrain, raphe, cerebellum and precerebellar system. This study represents the first detailed analysis of BCATc mRNA expression in the developing mouse brain.     

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain alpha-keto acid dehydrogenase (BCKD E1alpha subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1alpha or BCKD kinase, in liver BCKD E1alpha was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1alpha. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1alpha in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity.     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Sheep cytosolic branched-chain amino acid aminotransferase: cDNA cloning, primary structure and molecular modelling and its unique expression in muscles

This paper presents the cloning and the molecular modelling of the cytosolic branched-chain amino acid aminotransferase (BCATc) from sheep brain. The sheep BCATc cDNA (3 kb) encodes a mature polypeptide of 385 amino acids with a calculated molecular mass of 43072.93 Da. The sequence of the sheep BCATc cDNA is more similar to other mammalian BCATc cDNAs (53-87% identical) than to the sheep mitochondrial branched-chain amino acid aminotransferase (52%). Sheep BCATc belongs to the IV Folding class of pyridoxal-5'-phosphate-depending enzymes. Based on the known structure of the branched-chain amino acid aminotransferase (BCAT) from Escherichia coli, a molecular model of sheep BCATc (amino acid residues 62-385) was built. This is the first three-dimensional model of any mammalian BCAT. It suggests that the enzymatic mechanism of sheep BCATc and likely of all mammalian BCAT is very similar to the mechanism of the E. coli BCAT and confirms the hypotheses regarding to the substrate binding sites of E. coli BCAT. Sheep skeletal muscle, which is the main in vivo site for transamination of branched-chain amino acids, exhibits an unique expression of BCATc.     

Mitochondrial branched chain aminotransferase gene expression in AS-30D hepatoma rat cells and during liver regeneration after partial hepatectomy in rat

Branched chain aminotransferase (BCAT) is the first enzyme in the catabolism of branched chain amino acids (BCAA). Unlike other amino acid degrading enzymes present in liver, BCAT is only expressed in extrahepatic tissues, and is not regulated by dietary protein, glucagon or glucocorticoids. However, the mitochondrial (m) isoform of BCAT is highly expressed in the fetal liver and rapidly decays after birth. The purpose of the present work was to establish if liver cells under conditions of rapid cell proliferation such as in hepatoma AS30D cells or during liver regeneration after partial hepatectomy were associated with an increase in the activity and expression of BCATm. BCAT activity in mitochondria of AS30D cells was 18.6 mU/mg protein. Western, Northern blot, and immunohistochemical analysis revealed that AS30D hepatoma cells expressed only BCATm. The apparent Km of BCATm in isolated AS30D cells mitochondria for leucine, isoleucine and valine was 1.0+/-0.02, 1.3+/-0.1 and 2.1+/-0.1 mM, respectively. The regenerated liver showed BCAT activity from day 3 to day 6, and the maximal BCAT activity (7.0 mU/mg protein) was on day 5. By day 14 after partial hepatectomy BCAT activity and expression was almost undetectable. Interestingly, there was a relationship between BCAT activity and the Mr. of the immunoreactive band of BCATm. The presence of a 41 kDa band was associated with BCAT activity, whereas the 43 kDa band with undetectable activity. The results of this study indicate that BCATm activity is required in liver cells under conditions of rapid cell proliferation.     

Insulin increases branched-chain alpha-ketoacid dehydrogenase kinase expression in Clone 9 rat cells

The branched-chain amino acids (BCAA) are committed to catabolism by the activity of the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. BCKD activity is regulated through the action of the complex-specific BCKD kinase that phosphorylates two serine residues in the E1alpha subunit. Greater BCKD kinase expression levels result in a lower activity state of BCKD and thus a decreased rate of BCAA catabolism. Activity state varies among tissues and can be altered by diet, exercise, hormones, and disease state. Within individual tissues, the concentration of BCKD kinase reflects the activity state of the BCKD complex. Here we investigated the effects of insulin, an important regulator of hepatic metabolic enzymes, on BCKD kinase expression in Clone 9 rat cells. Insulin effected a twofold increase in message levels and a twofold increase in BCKD kinase protein levels. The response was completely blocked by treatment with LY-294002 and partially blocked by rapamycin, thus demonstrating a dependence on phosphatidylinositol 3-kinase and mTOR function, respectively. These studies suggest that insulin acts to regulate BCAA catabolism through stimulation of BCKD kinase expression.     

Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cells

The branched-chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.     

Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis

Unlike other amino acids, the branched-chain amino acids (BCAAs) largely bypass first-pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched-chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver–skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected, and no growth rate or body composition differences were observed in the transgenic animals as compared to wild-type mice. Feeding the transgenic animals a high-fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism, nor did the high-fat diet cause elevation in plasma BCAAs. However, the high-fat-diet-fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity.     

Total branched-chain amino acids requirement in patients with maple syrup urine disease by use of indicator amino acid oxidation with L-[1-13C]phenylalanine

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defects in the mitochondrial multienzyme complex branched-chain alpha-keto acid dehydrogenase (BCKD; EC 1.2.4.4), responsible for the oxidative decarboxylation of the branched-chain ketoacids (BCKA) derived from the branched-chain amino acids (BCAA) leucine, valine, and isoleucine. Deficiency of the enzyme results in increased concentrations of the BCAA and BCKA in body cells and fluids. The treatment of the disease is aimed at keeping the concentration of BCAA below the toxic concentrations, primarily by dietary restriction of BCAA intake. The objective of this study was to determine the total BCAA requirements of patients with classical MSUD caused by marked deficiency of BCKD by use of the indicator amino acid oxidation (IAAO) technique. Five MSUD patients from the MSUD clinic of The Hospital for Sick Children participated in the study. Each was randomly assigned to different intakes of BCAA mixture (0, 20, 30, 50, 60, 70, 90, 110, and 130 mg.kg(-1).day(-1)), in which the relative proportion of BCAA was the same as that in egg protein. Total BCAA requirement was determined by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO(2). The mean total BCAA requirement was estimated using a two-phase linear regression crossover analysis, which showed that the mean total BCAA requirement was 45 mg.kg(-1).day(-1), with the safe level of intake (upper 95% confidence interval) at 62 mg.kg(-1).day(-1). This is the first time BCAA requirements in patients with MSUD have been determined directly.     

Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.     

Molecular identification of a further branched-chain aminotransferase 7 (BCAT7) in tomato plants

Although the branched-chain amino acids (BCAAs) are essential components of the mammalian diet, our current understanding of their metabolism in plants is still limited. It is however well known that the branched-chain amino acid transaminases (BCATs) play a crucial role in both the synthesis and degradation of the BCAAs leucine, isoleucine and valine. We previously characterized the BCAT gene family in tomato, revealing it to be highly diverse in subcellular localization, substrate preference, and expression. Here we performed further characterization of this family and provide evidence for the presence of another member, BCAT7. On mapping the chromosomal location of this enzyme, it was possible to define the exact chromosome map position of the gene. Although in Arabidopsis thaliana the AtBCAT7 has been considered a pseudo-gene, quantitative evaluation of the expression levels of this gene revealed that the expression profile of the BCAT7 in different tissues of tomato (Solanum lycopersicum cv. M82) plants is highly variable with the highest expression found in developed flowers. By using a C-terminal E-GFP gene fusion we demonstrate that the BCAT7 is extraplastidial and in combination with the kinetic characterization of BCAT7 our results suggest that it most likely operates in BCAA degradation in vivo and support our hypothesis of another functional member of BCAT family. The combined data presented are discussed within the context of BCAA metabolism and its functions in higher plants.     

Immunocytochemical localization of beta-methylcrotonyl-CoA carboxylase in astroglial cells and neurons in culture

Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.     

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

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 x mg(-1) 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.     

Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways

Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5′-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5′-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.     

Hepatic histidase and muscle branched chain aminotransferase gene expression in experimental nephrosis

Protein and amino acid metabolism is altered during nephrotic syndrome. However, the expression of the amino acid degrading enzymes has not been well studied. The objective of this work was to assess the expression of hepatic histidase (Hal) and skeletal muscle mitochondrial branched chain amino transferase (BCATm) in rats with experimental nephrotic syndrome induced by a single injection of puromycin aminonucleoside (150 mg/kg). Six days after the injection rats were killed and hepatic Hal and skeletal muscle BCATm activities were measured. Also, total mRNA from both tissues was isolated and Hal and BCATm mRNA expression were analyzed by Northern blot. Rats with NS showed a reduction in food intake with respect to the control group. Hepatic Hal activity increased significantly in nephrotic and pair fed rats by 59% compared to control group. This change in activity was associated with a corresponding increase in Hal mRNA abundance. On the other hand, skeletal muscle BCATm activity and mRNA abundance were similar in the three groups studied. These results suggest that the increase in Hal expression was associated with the reduced food intake and not to the NS. However, BCAT expression did not change indicating the importance of BCAA in body nitrogen conservation.     

Cytosolic and mitochondrial isoenzymes of branched-chain amino acid aminotransferase during development of the rat.

The isoenzymic forms of branched-chain amino acid aminotransferase in mitochondria of rat tissues were compared with the better-known cytosolic forms in order to find any regular pattern of expression of these isoenzymes during development. Mitochondria of all tissues examined except brain contained only a type-I isoenzyme differing from the cytosolic type-I isoenzyme in heat stability and activation by mercaptoethanol. Foetal and adult brain mitochondria contained isoenzymes type III as well as type I. The large excess of type-I isoenzyme in foetal liver was localized in mitochondria, apparently of haematopoietic cells. The activity of this isoenzyme declined precipitously (by 80%) from day 19 of gestation at the same period and rate as does the volume fraction of haematopoietic cells that are then leaving the liver. Cortisol treatment accelerated the loss of these cells, and proportionally accelerated loss of the mitochondrial isoenzyme I. A development succession of type-I isoenzyme by the unique type II of liver parenchymal cell cytosols could not be demonstrated, since small, about equal, amounts of types I and II were always present in cytosols of foetal and adult liver. Developmental succession of isoenzymes within tissues was limited to cytosols and was demonstrated by the presence of cytosolic isoenzyme III in foetal and newborn skeletal muscle and kidney, organs which contain only isoenzyme I in the adult.